Conjugate of a tubulysin analog with branched linkers

ABSTRACT

Described is a conjugation of a tubulysin analog compound to a cell-binding molecule with branched/side-chain linkers for having better delivery of the conjugate compound and targeted treatment of abnormal cells. Also described are a branched-linkage method of conjugation of a tubulysin analog molecule to a cell-binding ligand, as well as methods of using the conjugate in targeted treatment of cancer, infection and autoimmune disease.

CROSS REFERENCE OF RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.16/759,175, filed on Apr. 24, 2020, entitled “A CONJUGATE OF A TUBULYSINANALOG WITH BRANCHED LINKERS,” which in turn is a national stageapplication of PCT/CN2017/120454, filed on Dec. 31, 2017. The entirecontent of each of the prior applications is hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to the conjugation of a tubulysin analogcompound to a cell-binding molecule with branched (side-chain) linkersfor having better pharmacokinetics in delivery of the conjugatecompound, resulting in much precise targeted treatment of abnormalcells. It also relates to a branched-linkage method of conjugation of atubulysin analog molecule to a cell-binding ligand, as well as methodsof using the conjugate in targeted treatment of cancer, infection andautoimmune disease.

BACKGROUND OF THE INVENTION

Antibody-drug conjugates (ADCs) have become one of promising targetingtherapies for cancer as evidenced by the clinical success of brentuximabvedotin (Adcetris) for relapsed/refractory Hodgkin lymphoma (Okeley, N.,et al, Hematol Oncol. Clin. North. Am, 2014, 28, 13-25; Gopal, A., etal, Blood 2015, 125, 1236-43) and ado-trastuzumab emtansine for relapsedHER2+ breast cancer (Peddi, P. and Hurvitz, S., Ther. Adv. Med. Oncol.2014, 6(5), 202-9; Lambert, J. and Chari, R., J. Med. Chem. 2014, 57,6949-64). The three important components, monoclonal antibody, cytotoxicpayload, and conditional linker of ADCs plus the sites where to link thelinker-payload components are all important factors to make success ofADC (L. Ducry and B Stump, Bioconjugate Chem., 2010, 21, 5-13; G. S.Hamilton, Biologicals 2015, 43, 318-32). It has be three decades tostudy each factor of the components of ADCs. However, linkertechnologies remain limited in scope, since drugs that are conjugatedmust contain certain reactive functional groups, ensure circulationstability, and facile drug release upon antigen binding andintracellular uptake, and importantly be not harming normal tissues oncethe linker-payload components are off-targeted during the circulation(Ponte, J. et al., Bioconj. Chem., 2016, 27(7), 1588-98; Dovgan, I., etal. Sci. Rep. 2016, 6, 30835; Ross, P. L. and Wolfe, J. L. J. Pharm.Sci. 105(2), 391-7; Chen, T. et al. J. Pharm. Biomed. Anal., 2016, 117,304-10).

In early ADCs, the linkers which were particularly used for ADCstargeting of liquid tumor were too labile, and led to the release offree drug in the circulation and consequent off-target toxicity (Bander,N. H. et al, Clin. Adv. Hematol. Oncol., 2012, 10, 1-16). In the currentgeneration of ADCs, the linkers are more stable, and the cytotoxicagents are significantly more potent (Behrens, C. R. and Liu, B., mAbs,2014. 6, 46-53). However, the off-target toxicity so far is still themajor challenge in development of ADC drugs (Roberts, S. A. et al,Regul. Toxicol. Pharmacol. 2013, 67, 382-91). For instance, in clinicalpractice Ado-trastuzumab emtansine (T-DM1, Kadcyla®) which is usedstable (none-cleavable) MCC linker has shown great benefit to patientswho have HER2-positive metastatic breast cancer (mBC) or who havealready been treated for mBC or developed HER2 tumor recurrence withinsix months of adjuvant therapy (Peddi, P. and Hurvitz, S., Ther. Adv.Med. Oncol. 2014, 6(5), 202-209; Piwko C, et al, Clin Drug Investig.2015, 35(8), 487-93; Lambert, J. and Chari, R., J. Med. Chem. 2014, 57,6949-64). But, T-DM1 had failed in clinic trial as first-line treatmentfor patients with HER2 positive unresectable locally advanced ormetastatic breast cancer and as the second line treatment ofHER2-positive advanced gastric cancer due to a little benefit topatients when comparison the side toxicity to the efficacy (Ellis, P.A., et al, J. Clin. Oncol. 2015, 33, (suppl; abstr 507 of 2015 ASCOAnnual Meeting); Shen, K. et al, Sci Rep. 2016; 6: 23262; de Goeij, B.E. and Lambert, J. M. Curr Opin Immunol 2016, 40, 14-23; Barrios, C. H.et al, J Clin Oncol 2016, 34, (suppl; abstr 593 of 2016 ASCO AnnualMeeting).

To address issues of the off-target toxicity, research and developmentinto ADC chemistry and design are now expanding the scopes of thelinker-payload compartments and conjugate chemistry beyond the solepotent payloads, and especially to address activity of thelinker-payload of ADCs toward targets/target diseases (Lambert, J. M.Ther Deliv 2016, 7, 279-82; Zhao, R. Y. et al, 2011, J. Med. Chem. 54,3606-23). Nowadays many drug developers and academic institutions arehighly focusing on establishing novel reliable specific conjugationlinkers and methods for site-specific ADC conjugation, which seem tohave longer circulation half-life, higher efficacy, potentiallydecreased off-target toxicity, and a narrow range of in vivopharmacokinetic (PK) properties of ADCs as well as better batch-to-batchconsistency in ADC production (Hamblett, K. J. et al, Clin. Cancer Res.2004, 10, 7063-70; Adem, Y. T. et al, Bioconjugate Chem. 2014, 25,656-664; Boylan, N. J. Bioconjugate Chem. 2013, 24, 1008-1016; Strop,P., et al 2013 Chem. Biol. 20, 161-67; Wakankar, A. mAbs, 2011, 3,161-172). These specific conjugation methods reported so far includeincorporation of engineered cysteines (Junutula, J. R. et al. Nat.Biotechnol. 2008, 26, 925-32; Junutula, J. R., et al 2010 Clin. CancerRes. 16, 4769; U.S. Pat. Nos. 8,309,300; 7,855,275; 7,521,541;7,723,485, WO2008/141044), selenocysteines (Hofer, T., et al.Biochemistry 2009, 48, 12047-57; Li, X., et al. Methods 2014, 65, 133-8;U.S. Pat. No. 8,916,159 for US National Cancer Institute), cysteinecontaining tag with perfluoroaromatic reagents (Zhang, C. et al. Nat.Chem. 2015, 8, 1-9), thiolfucose (Okeley, N. M., et al 2013 BioconjugateChem. 24, 1650), non-natural amino acids (Axup, J. Y., et al, Proc. Nat.Acad. Sci. USA. 2012, 109, 16101-6; Zimmerman, E. S., et al., 2014,Bioconjug. Chem. 25, 351-361; Wu, P., et al, 2009 Proc. Natl. Acad. Sci.106, 3000-5; Rabuka, D., et al, Nat. Protoc. 2012, 7, 1052-67; U.S. Pat.No. 8,778,631 and US Pat Appl. 20100184135, WO2010/081 110 for SutroBiopharma; WO2006/069246, 2007/059312, U.S. Pat. Nos. 7,332,571,7,696,312, and 7,638,299 for Ambrx; WO2007/130453, U.S. Pat. Nos.7,632,492 and 7,829,659 for Allozyne), conjugation to reducedintermolecular disulfides by re-bridging dibromomalemides (Jones, M. W.et al. J. Am. Chem. Soc. 2012, 134, 1847-52), bis-sulfone reagents(Badescu, G. et al. Bioconjug. Chem. 2014, 25, 1124-36; WO2013/190272,WO2014/064424 for PolyTherics Ltd), dibromopyridazinediones (Maruani, A.et al. Nat. Commun. 2015, 6, 6645), galactosyl- and sialyltransferases(Zhou, Q. et al. Bioconjug. Chem. 2014, 25, 510-520; US Pat Appl20140294867 for Sanofi-Genzyme), formylglycine generating enzyme (FGE)(Drake, P. M. et al. Bioconj. Chem. 2014, 25, 1331-41; Carrico, I. S. etal U.S. Pat. Nos. 7,985,783; 8,097,701; 8,349,910, and US Pat Appl20140141025, 20100210543 for Redwood Bioscience), phosphopantetheinyltransferases (PPTases) (Grunewald, J. et al. Bioconjug. Chem. 2015, 26,2554-62), sortase A (Beerli, R. R., et al. PLoS One 2015, 10, e0131177),genetically introduced glutamine tag with Streptoverticillium mobaraensetransglutaminase (mTG) (Strop, P., Bioconj. Chem., 2014, 25, 855-62;Strop, P., et al., Chem. Biol. 2013, 20, 161-7; U.S. Pat. No. 8,871,908for Rinat-Pfizer) or with microbial transglutaminase (MTGase) (Dennler,P., et al, 2014, Bioconjug. Chem. 25, 569-78; Siegmund, V. et al. Angew.Chemie-Int. Ed. 2015, 54, 13420-4; US pat appl 20130189287 for InnatePharma; U.S. Pat. No. 7,893,019 for Bio-Ker S.r.l. (IT)), anenzyme/bacterium forming an isopeptide bond-peptide bonds that formoutside of the protein main chain (Kang, H. J., et al. Science 2007,318, 1625-8; Zakeri, B. et al. Proc. Natl. Acad. Sci. USA 2012, 109,E690-7; Zakeri, B. & Howarth, M. J. Am. Chem. Soc. 2010, 132, 4526-7).

We have disclosed several conjugation methods of rebridging a pair ofthiols of the reduced inter chain disulfide bonds of a native antibody,such as using bromo maleimide and dibromomaleimide linkers(WO2014/009774), 2,3-disubstituted succinic/2-monosubstituted/2,3-disubstituted fumaric or maleic linkers (WO2015/155753,WO20160596228), acetylenedicarboxylic linkers (WO2015/151080,WO20160596228) or hydrazine linkers (WO2015/151081). The ADCs made withthese linkers and methods have demonstrated better therapeutic indexwindows than the traditionally unselective conjugation via the cysteineor lysine residues on an antibody. Here we disclose the invention oftubulysin conjugate containing a long side chain linker. The long sidechain linker can prevent an antibody-drug conjugate from hydrolysis by ahydrolase, e.g. a proteinase or an esterase and lead to the conjugatemore stable in the circulation.

Tubulysins as a potent cytotoxic agents are well known in the art andcan be isolated from natural sources according to known methods orprepared synthetically according to known methods (e.g. Balasubramanian,R., et al. J. Med. Chem., 2009, 52, 238-40; Wipf, P., et al. Org. Lett.,2004, 6, 4057-60; Pando, O., et al. J. Am. Chem. Soc., 2011, 133,7692-5; Reddy, J. A., et al. Mol. Pharmaceutics, 2009, 6, 1518-25;Raghavan, B., et al. J. Med. Chem., 2008, 51, 1530-33; Patterson, A. W.,et al. J. Org. Chem., 2008, 73, 4362-9; Pando, O., et al. Org. Lett.,2009, 11 (24), 5567-9; Wipf, P., et al. Org. Lett., 2007, 9 (8), 1605-7;Friestad, G. K., Org. Lett.,2004, 6, 3249-52; Peltier, H. M., et al. J.Am. Chem. Soc., 2006, 128, 16018-9; Chandrasekhar, S., et al J. Org.Chem., 2009, 74, 9531-4; Liu, Y., et al. Mol. Pharmaceutics, 2012, 9,168-75; Friestad, G. K., et al. Org. Lett., 2009, 11, 1095-8; Kubicek,K., et al., Angew Chem Int Ed Engl, 2010.49: 4809-12; Chai, Y., et al.,Chem Biol, 2010, 17: 296-309; Ullrich, A., et al., Angew Chem Int EdEngl, 2009, 48, 4422-5; Sani, M., et al. Angew Chem Int Ed Engl, 2007,46, 3526-9; Domling, A., et al., Angew Chem Int Ed Engl, 2006, 45,7235-9; Patent applications: Zanda, M., et al, Can. Pat. Appl. CA2710693 (2011); Chai, Y., et al. Eur. Pat. Appl. 2174947 (2010), WO2010034724; Leamon, C. et al, WO2010033733, WO 2009002993; Ellman, J.,et al, PCT WO2009134279; WO 2009012958, US appl. 20110263650,20110021568; Matschiner, G., et al, WO2009095447; Vlahov, I., et al,WO2009055562, WO 2008112873; Low, P., et al, WO2009026177; Richter, W.,WO2008138561; Kjems, J., et al, WO 2008125116; Davis, M.; et al,WO2008076333; Diener, J.; et al, U.S. Pat.Appl. 20070041901,WO2006096754; Matschiner, G., et al, WO2006056464; Vaghefi, F., et al,WO2006033913; Doemling, A., Ger. Offen. DE102004030227, WO2004005327,WO2004005326, WO2004005269; Stanton, M., et al, U.S. Pat. Appl. Publ.20040249130; Hoefle, G., et al, Ger. Offen. DE10254439, DE10241152,DE10008089; Leung, D., et al, WO2002077036; Reichenbach, H., et al, Ger.Offen. DE19638870; Wolfgang, R., US20120129779; Chen, H., US appl.20110027274. We previously disclosed the construction of tubulysinsconjugate (PCT/IB2012/053554) for targeted treatment of cancer,infection and autoimmune disease. The present invention of tubulysinconjugate containing a long branched (side-chain) linker can prolong thehalf-life of a conjugate during the targeted delivery and minimizeexposure to non-target cells, tissues or organs during the bloodcirculation, resulting in less the off-target toxicity.

SUMMARY OF THE INVENTION

The present invention provides branched-linkage of a tubulysin analog toan antibody. It also provides a method of conjugation of a tubulysinanalog to an antibody with the side chain-linker.

In one aspect of the present invention, a conjugate containing a sidechain-linkage is represented by Formula (I):

wherein

“

” represents a single bond; n is 1 to 30;

T is a cell-binding agent/molecule, selected from the group consistingof an antibody, a single chain antibody, an antibody fragment that bindsto a target cell, a monoclonal antibody, a single chain monoclonalantibody, a monoclonal antibody fragment that binds to the target cell,a chimeric antibody, a chimeric antibody fragment that binds to thetarget cell, a domain antibody, a domain antibody fragment that binds tothe target cell, an adnectin that mimics antibody, DARPins, alymphokine, a hormone, a vitamin, a growth factor, a colony stimulatingfactor, a nutrient-transport molecule (a transferrin), and a bindingpeptide, protein, small molecule attached on albumin, a polymer, adendrimer, a liposome, a nanoparticle, a vesicle, or a (viral) capsid;

L₁ and L₂ are a chain of atoms selected from C, N, O, S, Si, and P,preferably having 0-500 atoms, which covalently connects to W and V₁,and V₁ and V₂. The atoms used in forming the L₁ and L₂ may be combinedin all chemically relevant ways, such as forming alkylene, alkenylene,and alkynylene, ethers, polyoxyalkylene, esters, amines, imines,polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides,carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids,peptides, acyloxylamines, hydroxamic acids, or combination abovethereof. Preferably L₁ and L₂ are, the same or different, independentlyselected from O, NH, N, S, P, NNH, NHNH, N(R₃), N(R₃)N(R₃′), CH, CO,C(O)NH, C(O)O, NHC(O)NH, NHC(O)O, polyethyleneoxy unit of formula(OCH₂CH₂)_(p)OR₃, or (OCH₂CH—(CH₃))_(p)OR₃, or NH(CH₂CH₂O)_(p)R₃, orNH(CH₂CH(CH₃)O)_(p)R₃, or N[(CH₂CH₂O)_(p)R₃]-[(CH₂CH₂O)_(p)′R₃′], or(OCH₂CH₂)_(p)COOR₃, or CH₂CH₂(OCH₂CH₂)_(p)COOR₃, wherein p and p′ areindependently an integer selected from 0 to about 1000, or combinationthereof, C₁-C₈ of alkyl; C₂-C₈ of heteroalkyl, alkylcycloalkyl,heterocycloalkyl; C₃-C₈ of aryl, Ar-alkyl, heterocyclic, carbocyclic,cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or(Aa)_(r), r=1-12(one to 12 amino acid units), which is composed fromnatural or unnatural amino acids, or the same or different sequences ofdipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide,heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide ordodecapeptide unit;

W is a stretcher unit, normally a self-immolative spacer, a peptidicunit, a hydrazone, a disulfide, a thioether, an ester, or an amide bond;w is 1 or 2 or 3;

V₁ and V₂ are independently a spacer unit and selected from O, NH, S,C₁-C₈ alkyl, C₂-C₈ heteroalkyl, alkenyl, or alkynyl, C₃-C₈ aryl,heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, oralkylcarbonyl, or (Aa)_(r), r=1-12(one to 12 amino acid units), which iscomposed from a natural or unnatural amino acid, or the same ordifferent sequences of dipeptide, tripeptide, tetrapeptide,pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide,decapeptide, undecapeptide or dodecapeptide unit; or (CH₂CH₂O)_(p), p is0-1000; and v₁ and v₂ are independently 0, 1 or 2, but v₁ and v₂ are 0at the same time; when v₁ or v₂ is 0, it means that one of the sidechain Q₁ or Q₂ fragment is absent.

Q₁ and Q₂ are independently represented by Formula (I-q1):

wherein

is the site linked to L₁ or L₂, G₁ and G₂ are independently OC(O),NHC(O), C(O), CH₂, NH, OC(O)NH, NHC(O)NH, O, S, B, P(O)(OH), NHP(O)(OH),NHP(O)(OH)NH, CH₂P(O)(OH)NH, OP(O)(OH)O, CH₂P(O)(OH)O, NHS(O)₂,NHS(O)₂NH, CH₂S(O)₂NH, OS(O)₂O, CH₂S(O)₂O, Ar, ArCH₂, ArO, ArNH, ArS,ArNR₁, (Aa)_(r), (r=1-12); X₁ and X₂ are independently O, CH₂, S, NH,N(R₁), ⁺NH(R₁), +N(R₁)(R₂), C(O), OC(O), OC(O)O, OC(O)NH, NHC(O)NH; Y₂is O, NH, NR₁, CH₂, S. Ar; G₃ is OH, SH, OR₁, SR₁, OC(O)R₁, NHC(O)R₁,C(O)R₁, CH₃, NH₂, NR₁, ⁺NH(R₁), ⁺N(R₁)(R₂), C(O)OH, C(O)NH₂, NHC(O)NH₂,BH₂, BR₁R₂, P(O)(OH)₂, NHP(O)(OH)₂, NHP(O)(NH₂)₂, S(O)₂(OH),(CH₂)_(q1)C(O)OH, (CH₂)_(q1)P(O)(OH)₂, C(O)(CH₂)_(q1)C(O)OH,OC(O)(CH₂)_(q1)C(O)OH, NHC(O)(CH₂)_(q1)C(O)OH, CO(CH₂)_(q1)P(O)(OH)₂,NHC(O)O(CH₂)_(q1)C(O)OH, OC(O)NH(CH₂)_(q1)C(O)OH,NHCO(CH₂)_(q1)P(O)(OH)₂, NHC(O)(NH)(CH₂)_(q1)C(O)OH,CONH(CH₂)_(q1)P(O)(OH)₂, NHS(O)₂(CH₂)_(q1)C(O)OH, CO(CH₂)_(q1)S(O)₂(OH),NHS(O)₂NH(CH₂)_(q1)C(O)OH, OS(O)₂NH(CH₂)_(q1)C(O)OH,NHCO(CH₂)_(q1)S(O)₂(OH), NHP(O)(OH)(NH)(CH₂)_(q1)C(O)OH,CONH(CH₂)_(q1)S(O)(OH), OP(O)(OH)₂, (CH₂)_(q1)P(O)(NH)₂, NHS(O)₂(OH),NHS(O)₂NH₂, CH₂S(O)₂NH₂, OS(O)₂OH, OS(O)₂OR₁, CH₂S(O)₂OR₁, Ar, ArR₁,ArOH, ArNH₂, ArSH, ArNHR₁, or (Aa)_(q1); p₁, p₂ and p₃ are independently0-100 but are not 0 at the same time; q₁ and q₂ are independently 0-24;

Preferably Q₁ and Q₂ are independently a C₂-C₉₀ polycarboxylacid or aC₂-C₉₀ polyalkylamine, a C₆-C₉₀ oligosaccharide or polysaccharide, aC₆-C₉₀ zwitterionic betaines or zwitterionic poly(sulfobetaine)) (PSB)sthat consist of a quaternary ammonium cation and a sulfonate anion,biodegradable polymer (such as composed of poly (lactic/glycolic) acid(PLGA), poly(acrylates), chitosan, copolymer ofN-(2-hydroxypropyl)methacrylamide, poly[2-(methacryloyloxy)ethylphosphorylcholine] (PMPC), poly-L-glutamic acid,poly(lactide-co-glycolide) (PLG), poly(lactide-co-glycolide),Poly(ethylene glycol)(PEG), poly(propylene glycol)(PPG),poly(lactide-co-glycolide), poly(ethylene glycol)-modified peptides,poly(ethylene glycol)-modified lipids, poly(ethylene glycol)-modifiedalkylcarboxic acid, poly(ethylene glycol)-modified alkylamine,poly(lactide-co-glycolide, hyaluronic acid (HA) (glycosaminoglycan),heparin/heparan sulfate (HSGAGs), chondroitin sulfate/dermatan sulfate(CSGAGs), poly(ethylene glycol)-modified alkylsulfate, poly(ethyleneglycol)-modified alkylphosphate, or poly(ethylene glycol)-modified alkylquaternary ammonium; D is tubulysin analog having the following formula(II):

or a pharmaceutically acceptable salt, hydrates, or hydrated salt; or apolymorphic crystalline structure; or an optical isomer, racemate,diastereomer or enantiomer thereof,

wherein

is a linkage site that links to W independently;

wherein R¹, R², R³, and R⁴ are independently H, C₁˜C₈ alkyl; C₂˜C₈heteroalkyl, or heterocyclic; C₃˜C₈ aryl, Ar-alkyl, cycloalkyl,alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbocyclic,or alkylcarbonyl; or R¹R², R¹R³, R²R³, R³R⁴, R⁵R⁶, R¹¹R¹² or R¹³R¹⁴ forma 3˜7 membered carbocyclic, cycloalkyl, heterocyclic, heterocycloalkyl,aromatic or heteroaromatic ring system; R¹ and R² can be independentlyabsent when they link to W independently or simultaneously, Y¹ is N orCH;

wherein R⁵, R⁶, R⁸, R¹⁰ and R¹¹ are independently H, or C₁˜C₄ alkyl orheteroalkyl;

wherein R⁷ is independently H, R¹⁴, —R¹⁴C(═O)X¹R¹⁵; or —R¹⁴X¹R¹⁵; X¹ isO, S, S—S, NH, CH₂ or NR¹⁴;

wherein R⁹ is selected from H, OH, —O—, ═O, —OR¹⁴, —OC(═O)R¹⁴,—OC(═O)NHR¹⁴—, —OC(═O) R¹⁴SSR¹⁵—, OP(═O)(OR¹⁴)—, —OC(═O)NR¹⁴R¹⁵,OP(═O)(OR¹⁴), or OR¹⁴OP(═O)(OR¹⁵);

wherein R¹¹ is independently H, R¹⁴, —R¹⁴C(═O)R¹⁶, —R¹⁴X²R¹⁶,—R¹⁴C(═O)X², wherein X² is —O—, —S—, —NH—, —N(R¹⁴)—, —O—R¹⁴—, —S—R¹⁴—,—S(═O)—R¹⁴—, or —NHR¹⁴;

wherein R¹² is R¹⁵,—OH, —SH, —NH₂, NH, NHNH₂, —NH(R¹⁵), —OR¹⁵,—R¹⁵COR¹⁶, —R¹⁵COOR¹⁶, —R¹⁵C(O)NH₂, —R¹⁵C(O)NHR¹⁷, —SR¹⁶, R¹⁵S(═O)R¹⁶, —R¹⁵P(═O)(OR¹⁷)₂, —R¹⁵OP(═O)(OR¹⁷)₂, —CH₂OP(═O)(OR¹⁷)₂, —R¹⁵SO₂R¹⁷,—R¹⁵X²R¹⁶, —R¹⁵C(═O)X², where X² is —O—, OH, SH, —S—, NH₂, —NH—,—N(R¹⁵)—, —O—R¹⁵—, —S—R¹⁵—, —S(═O)—R¹⁵—, CH₂ or—NHR¹⁵—;

R¹³ and R¹⁴ are independently H, O, S, NH, N(R¹⁵), NHNH, —OH, —SH, —NH₂,NH, NHNH₂, —NH(R¹⁵), —OR¹⁵, CO, —COX², —COX²R¹⁶, R¹⁷, F, Cl, Br, I,SR¹⁶, NR¹⁶R¹⁷, N═NR¹⁶, N═R¹⁶, NO₂, SOR¹⁶R¹⁷, SO₂R¹⁶, SO₃R¹⁶, R¹⁶, R¹⁷,POR¹⁶R¹⁷, PO₂R¹⁶R¹⁷, OP(O)(OR¹⁷)₂, OCH₂OP(O)(OR¹⁷)₂, OC(O)R¹⁷,OC(O)OP(O)(OR¹⁷)₂, PO(OR¹⁶)(OR¹⁷), OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂, OC(O)NHR¹⁷;—O—(C₄-C₁₂ glycoside), -N-(C₄-C₁₂ glycoside); C₁˜C₈ alkyl, heteroalkyl;C₂-C₈ of alkenyl, alkynyl, heteroalkyl, heterocycloalkyl; C₃-C₈ of aryl,Ar-alkyl, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl,heteroaryl, or 2-8 carbon atoms of esters, ether, or amide; or peptidescontaining 1-8 amino acids (NH(Aa)_(1˜8) or CO(Aa)_(1˜8) (N-terminal orC-terminal 1-8 the same or different amino acids), or polyethyleneoxyunit of formula (OCH₂CH₂)_(p) or (OCH₂CH(CH₃))_(p), wherein p is aninteger from 0 to about 1000, or combination of above groups thereof; X²is O, S, S—S, NH, CH₂, OH, SH, NH₂, CHR¹⁴ or NR¹⁴;

R¹⁵R¹⁶ and R¹⁷ is independently H, C₁˜C₈ alkyl, heteroalkyl; C₂-C₈ ofalkenyl, alkynyl, heteroalkyl, heterocycloalkyl; C₃-C₈ of aryl,Ar-alkyl, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl,heteroaryl, alkylcarbonyl, or Na⁺, K⁺, Cs⁺, Li⁺, Ca²⁺, Mg⁺, Zn²⁺,N⁺(R¹)(R²)(R³) (R⁴), HN⁺(C₂H₅OH)₃ salt;

Y¹ and Y² are independently N or CH; q is 0 or 1; when q=0, Y³ does notexist, Y⁴, Y⁵, Y⁶ and Y⁷ are independently CH, N, NH, O, S, or N (R1),thus Y², Y⁴ Y⁵ Y⁶ and Y⁷form a heteroaromatic ring of furan, pyrrolethiophene, thiazole, oxazole and imidazole, pyrazole, triazole,tetrazole, thiadiazole; when q=1, Y³, Y⁴, Y⁵, Y⁶ and Y⁷ areindependently CH or N, thus Y², Y³, Y⁴, Y⁵, Y⁶ and Y⁷ form aromatic ringof benzene, pyridine, pyridazine, pyrimidine, pyrazine, triazine,tetrazine, pentazine;

In another aspect of the present invention, a conjugate containing aside chain-linkage is represented by Formula (III):

wherein D, W, w, L₁, L₂, Q₁, Q₂, V₁, V₂, v₁, v₂, n, T are defined thesame as in Formula (I).

In another aspect of the present invention, the side chain-linkagecompound is represented by Formula (IV), which can readily react to acell-binding molecule T to form a conjugate of Formula (I):

wherein D, W, w, L₁, L₂, Q₁, Q₂, V₁, V₂, v₁, v₂, and n, are defined thesame as in Formula (I); Lv1 is a function group described below.

In another aspect of the present invention, the side chain-linkagecompound is represented by Formula (V), which can readily react to acell-binding molecule T to form a conjugate of Formula (III):

wherein D, W, w, L₁, L₂, Q₁, Q₂, V₁, V₂, v₁, v₂, and n, are defined thesame as in Formula (I).

L_(V1) and L_(V2) represent the same or different reacting group thatcan be reacted with a thiol, amine, carboxylic acid, selenol, phenol orhydroxyl group on a cell-binding molecule. L_(V1) and L_(V2) areindependently selected from OH; F; Cl; Br; I; nitrophenol;N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol;tetrafluorophenol; difluorophenol; mono-fluorophenol; pentachlorophenol;triflate;imidazole;dichlorophenol;tetrachlorophenol;1-hydroxybenzotriazole;tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3′-sulfonate,anhydridesformed its self, or formed with the other anhydride, e.g. acetylanhydride, formyl anhydride; or an intermediate molecule generated witha condensation reagent for peptide coupling reactions, or for Mitsunobureactions. The examples of condensation reagents are: EDC(N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide), DCC(Dicyclohexyl-carbodiimide), N,N′-Diisopropylcarbodiimide (DIC),N-Cyclohexyl-N′-(2-morpholino-ethyl)carbodiimidemetho-p-toluenesulfonate (CMC,or CME-CDI), 1,1′-Carbonyldiimi-dazole(CDI), TBTU (O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate),N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)-uroniumhexafluoro-phosphate (HBTU),(Benzotriazol-1-yloxy)tris(dimethylamino)-phosphoniumhexafluorophosphate (BOP),(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate(PyBOP), Diethyl cyanophosphonate (DEPC),Chloro-N,N,N′,N′-tetramethylformamidiniumhexafluorophosphate,1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU),1-[(Dimethylami-no)(morpholino)methylene]-1H-[1,2,3]triazolo[4,5-b]pyridine-1-ium3-oxide hexafluoro-phosphate (HDMA),2-Chloro-1,3-dimethyl-imidazolidinium hexafluorophosphate (CIP),Chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP),Fluoro-N,N,N′,N′-bis(tetramethylene)formamidinium hexafluorophosphate(BTFFH), N,N,N′,N′-Tetramethyl-S-(1-oxido-2-pyridyl)thiuroniumhexafluorophosphate, O-(2-Oxo-1(2H)pyridyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TPTU),S-(1-Oxido-2-pyridyl)-N,N,N′,N′-tetramethylthiuronium tetrafluoroborate,O-[(Ethoxycarbonyl)-cyanomethylenamino]-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HOTU), (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU),O-(Benzotriazol-1-yl)-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate (HBPyU), N-Benzyl-N′-cyclohexyl-carbodiimide (with,or without polymer-bound), Dipyrrolidino(N-succinimidyl-oxy)carbeniumhexafluoro-phosphate (HSPyU), Chlorodipyrrolidinocarbeniumhexafluorophosphate (PyClU), 2-Chloro-1,3-dimethylimidazolidiniumtetrafluoroborate(CIB), (Benzotriazol-1-yloxy)dipiperidino-carbeniumhexafluorophosphate (HBPipU),O-(6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TCTU), Bromotris(dimethylamino)-phosphoniumhexafluorophosphate (BroP), Propylphosphonic anhydride (PPACA, T3P®),2-Morpholinoethyl isocyanide (MEI),N,N,N′,N′-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate(HSTU), 2-Bromo-1-ethyl-pyridinium tetrafluoroborate (BEP),O-[(Ethoxycarbonyl)cyano-methylenamino]-N,N,N′,N′-tetra-methyluroniumtetrafluoroborate (TOTU),4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholiniumchloride (MMTM,DMTMM), N,N,N′,N′-Tetramethyl-O-(N-succinimidyl)uroniumtetrafluoroborate (TSTU),O-(3,4-Dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoro-borate (TDBTU),1,1′-(Azodicarbonyl)-dipiperidine (ADD),Di-(4-chlorobenzyl)azodicarboxylate (DCAD), Di-tert-butylazodicarboxylate (DBAD),Diisopropyl azodicarboxylate (DIAD), Diethylazodicarboxylate (DEAD). In addition, L_(V1) and L_(V2) can be ananhydride, formed by acid themselves or formed with other C₁˜C₈ acidanhydrides;

The present invention further relates to a method of making acell-binding molecule-drug conjugate of Formula (I) and Formula (III) aswell the application of the conjugates of Formula (I) and Formula (III).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the general synthesis of Tuv component of a Tubulysinanalog.

FIG. 2 shows the synthesis of tubulysin components.

FIG. 3 shows the synthesis of tubulysin components.

FIG. 4 shows the synthesis of components of tubulysin analogs.

FIG. 5 shows the synthesis of components of tubulysin analogs.

FIG. 6 shows the synthesis of components of tubulysin analogs.

FIG. 7 shows the synthesis of components of tubulysin analogs.

FIG. 8 shows the synthesis of components of tubulysin analogs containinga conjugate linker.

FIG. 9 shows the synthesis of components of tubulysin analogs and theirconjugations to an antibody.

FIG. 10 shows the synthesis of components of a side-chain linker.

FIG. 11 shows the synthesis of Tubulysin analogs containing a side-chainlinker and their conjugations to an antibody.

FIG. 12 shows the synthesis of Tubulysin analogs containing a side-chainlinker and their conjugations to an antibody.

FIG. 13 shows the synthesis of Tubulysin analogs containing a side-chainlinker and their conjugations to an antibody.

FIG. 14 shows the synthesis of components of tubulysin analogscontaining a side-chain linker and their conjugations to an antibody.

FIG. 15 shows the synthesis of components of a side-chain linker andtheir linkage to a Tup component.

FIG. 16 shows the synthesis of Tubulysin analogs containing a side-chainlinker and their conjugations to an antibody.

FIG. 17 shows the synthesis of Tubulysin analogs containing a side-chainlinker and their conjugations to an antibody.

FIG. 18 shows the synthesis of components of tubulysin analogs.

FIG. 19 shows the synthesis of components of tubulysin analogs.

FIG. 20 shows the synthesis of Tubulysin analogs containing a side-chainlinker and their conjugations to an antibody.

FIG. 21 shows the synthesis of Tubulysin analogs containing a side-chainlinker and their conjugations to an antibody.

FIG. 22 shows the synthesis of Tubulysin analogs containing a side-chainlinker and their conjugations to an antibody.

FIG. 23 shows the synthesis of Tubulysin analogs containing a side-chainlinker and their conjugations to an antibody.

FIG. 24 shows the synthesis of Tubulysin analogs containing a side-chainlinker and their conjugations to an antibody.

FIG. 25 shows the synthesis of Tubulysin analogs containing a side-chainlinker and their conjugations to an antibody.

FIG. 26 shows the synthesis of Tubulysin components containing aside-chain linker.

FIG. 27 shows the synthesis of Tubulysin analogs containing a side-chainlinker and their conjugations to an antibody via a pair of thiols in theantibody.

FIG. 28 shows the synthesis of Tubulysin components containingside-chain linkers.

FIG. 29 shows the synthesis of Tubulysin analogs containing a side-chainlinker and their conjugations to an antibody via a pair of thiols in theantibody.

FIG. 30 shows the synthesis of Tubulysin analogs containing side-chainlinkers and their conjugations to an antibody via a pair of thiols inthe antibody.

FIG. 31 shows the synthesis of Tubulysin components.

FIG. 32 shows the synthesis of Tubulysin components.

FIG. 33 shows the synthesis of Tubulysin components.

FIG. 34 shows the synthesis of Tubulysin components containing aside-chain linker.

FIG. 35 shows the synthesis of a Tubulysin analog containing aside-chain linker and its conjugation to an antibody.

FIG. 36 shows the synthesis of a Tubulysin analog containing aside-chain linker and its conjugation to an antibody.

FIG. 37 shows the synthesis of a Tubulysin analog containing aside-chain linker and its conjugation to an antibody.

FIG. 38 shows the synthesis of a Tubulysin analog containing aside-chain linker and its conjugation to an antibody.

FIG. 39 shows the synthesis of a Tubulysin analog containing aside-chain linker and its conjugation to an antibody.

FIG. 40 shows the synthesis of a Tubulysin analog containing aside-chain linker and its conjugation to an antibody.

FIG. 41 shows the synthesis of a Tubulysin analog containing aside-chain linker and its conjugation to an antibody.

FIG. 42 shows the synthesis of a Tubulysin analog.

FIG. 43 shows the synthesis of a Tubulysin analog.

FIG. 44 shows the synthesis of a Tubulysin analog.

FIG. 45 shows the synthesis of a Tubulysin analog and its conjugation toan antibody.

FIG. 46 shows the synthesis of a Tubulysin analog and its conjugation toan antibody.

FIG. 47 shows the synthesis of a Tubulysin analog.

FIG. 48 shows the synthesis of a Tubulysin analog and its conjugation toan antibody.

FIG. 49 shows the synthesis of a Tubulysin analog and its conjugation toan antibody.

FIG. 50 shows the synthesis of a Tubulysin analog containing aside-chain linker and its conjugation to an antibody.

FIG. 51 shows the synthesis of a Tubulysin analog and its conjugation toan antibody.

FIG. 52 shows the synthesis of a Tubulysin analog containing aside-chain linker and its conjugation to an antibody.

FIG. 53 shows the synthesis of Tubulysin analogs containing side-chainlinkers and their conjugations to an antibody via a pair of thiols inthe antibody.

FIG. 54 shows the synthesis of a Tubulysin analog containing aside-chain linker and its conjugations to an antibody.

FIG. 55 shows the synthesis of a Tubulysin analog containing aside-chain linker to an antibody.

FIG. 56 shows the synthesis of a Tubulysin analog containing aside-chain linker and its conjugation to an antibody.

FIG. 57 shows the synthesis of a Tubulysin analog containing aside-chain linker.

FIG. 58 shows the synthesis of Tubulysin analogs containing side-chainlinkers and their conjugations to an antibody via a pair of thiols inthe antibody.

FIG. 59 shows the synthesis of Tubulysin analogs containing side-chainlinkers and their conjugations to an antibody via a pair of thiols inthe antibody.

FIG. 60 shows the synthesis of Tubulysin analogs containing side-chainlinkers and their conjugations to an antibody via a pair of thiols inthe antibody.

FIG. 61 shows the synthesis of a Tubulysin analog containing aside-chain linker and its conjugation to an antibody.

FIG. 62 shows the synthesis of a Tubulysin analog containing aside-chain linker and its conjugation to an antibody.

FIG. 63 shows the comparison of the anti-tumor effect of conjugatecompounds 474, 486, 493, 601, 626, 637, 641, 669, 673, 680, and 692 withT-DM1 using human gastric tumor N87 cell model, i.v., one injection atdosing of 6 mg/kg.

FIG. 64 shows the stability study of the conjugates (680 and 692) havinga side chain-linkage in comparison with T-DM1 and a regular conjugate(compound 133) having a mono-linkage in the mouse serum.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Alkyl” refers to an aliphatic hydrocarbon group or univalent groupsderived from alkane by removal of one or two hydrogen atoms from carbonatoms. It may be straight or branched having C₁-C₈ (1 to 8 carbon atoms)in the chain. “Branched” means that one or more lower C numbers of alkylgroups such as methyl, ethyl or propyl are attached to a linear alkylchain. Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl,n-butyl, t-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl,cyclohexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl,2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl,3-methyl-hexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl,3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl,n-heptyl, isoheptyl, n-octyl, and isooctyl. A C₁-C₈ alkyl group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —S(O)₂R′,—S(O)R′, —OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′) ₂ and —CN; where eachR′ is independently selected from —C₁-C₈ alkyl and aryl.

“Halogen” refers to fluorine, chlorine, bromine or iodine atom;preferably fluorine and chlorine atom.

“Heteroalkyl” refers to C₂-C₈ alkyl in which one to four carbon atomsare independently replaced with a heteroatom from the group consistingof O, S and N.

“Carbocycle” refers to a saturated or unsaturated ring having 3 to 8carbon atoms as a monocycle or 7 to 13 carbon atoms as a bicycle.Monocyclic carbocycles have 3 to 6 ring atoms, more typically 5 or 6ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, arranged as abicycle [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicycle [5,6] or [6,6]system. Representative C₃-C₈carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl,-cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl,-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl,and-cyclooctadienyl.

A “C₃-C₈ carbocycle” refers to a 3-, 4-, 5-, 6-, 7—or 8-memberedsaturated or unsaturated nonaromatic carbocyclic ring. A C₃-C₈carbocycle group can be unsubstituted or substituted with one or moregroups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl),-aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂,—NHC(O)R′, —SR′, —S(O)R′,—S(O)₂R′, —OH, -halogen, —N₃, —NH₂, —NH(R′),—N(R′) 2 and —CN; where each R′ is independently selected from —C₁-C₈alkyl and aryl.

“Alkenyl” refers to an aliphatic hydrocarbon group containing acarbon-carbon double bond which may be straight or branched having 2 to8 carbon atoms in the chain. Exemplary alkenyl groups include ethenyl,propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl,hexylenyl, heptenyl, octenyl.

“Alkynyl” refers to an aliphatic hydrocarbon group containing acarbon-carbon triple bond which may be straight or branched having 2 to8 carbon atoms in the chain. Exemplary alkynyl groups include ethynyl,propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5-pentynyl, n-pentynyl,hexylynyl, heptynyl, and octynyl.

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical of 1-18 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to: methylene (—CH₂—), 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkene. Typicalalkenylene radicals include, but are not limited to: 1,2-ethylene(—CH═CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne. Typicalalkynylene radicals include, but are not limited to: acetylene,propargyl and 4-pentynyl.

“Aryl” or Ar refers to an aromatic or hetero aromatic group, composed ofone or several rings, comprising three to fourteen carbon atoms,preferentially six to ten carbon atoms. The term of “hetero aromaticgroup” refers one or several carbon on aromatic group, preferentiallyone, two, three or four carbon atoms are replaced by O, N, Si, Se, P orS, preferentially by O, S, and N. The term aryl or Ar also refers to anaromatic group, wherein one or several H atoms are replacedindependently by -R′, -halogen, —OR′, or —SR′, —NR′R″, —N═NR′, —N═R′,—NR′R″,—NO₂, —S(O)R′, —S(O)₂R′, —S(O)₂OR′, —OS(O)₂OR′, —PR′R″,—P(O)R′R″, —P(OR′)(OR″), —P(O)(OR′)(OR″) or —OP(O)(OR′)(OR″) wherein R′,R¹¹ are independently H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl,arylalkyl, carbonyl, or pharmaceutical salts.

“Heterocycle” refers to a ring system in which one to four of the ringcarbon atoms are independently replaced with a heteroatom from the groupof O, N, S, Se, B, Si and P. Preferable heteroatoms are O, N and S.Heterocycles are also described in The Handbook of Chemistry andPhysics, 78th Edition, CRC Press, Inc., 1997-1998, p. 225 to 226, thedisclosure of which is hereby incorporated by reference. Preferrednonaromatic heterocyclic include epoxy, aziridinyl, thiiranyl,pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl,tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, dioxolanyl,piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl,pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl,thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, dihydropyranyl,tetrahydropyridyl, dihydropyridyl, tetrahydropyrimidinyl,dihydrothiopyranyl, azepanyl, as well as the fused systems resultingfrom the condensation with a phenyl group.

The term “heteroaryl” or aromatic heterocycles refers to a 3 to 14,preferably 5 to 10 membered aromatic hetero, mono-, bi-, or multi-cyclicring. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl,pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl,imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl,1,2,4-thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoquinolyl,benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl,isoxazolyl, pyridyl-N-oxide, as well as the fused systems resulting fromthe condensation with a phenyl group.

“Alkyl”, “cycloalkyl”, “alkenyl”, “alkynyl”, “aryl”, “heteroaryl”,“heterocyclic” and the like refer also to the corresponding “alkylene”,“cycloalkylene”, “alkenylene”, “alkynylene”,

“arylene”, “heteroarylene”, “heterocyclene” and the likes which areformed by the removal of two hydrogen atoms.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heteroaryl radical. Examples ofheteroarylalkyl groups are 2-benzimidazolylmethyl, 2-furylethyl.

Examples of a “hydroxyl protecting group” includes, methoxymethyl ether,2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether,p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether,triisopropylsilyl ether, t-butyldimethylsilyl ether,triphenylmethylsilyl ether, acetate ester, substituted acetate esters,pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Such leaving groups are well known in the art,and examples include, a halide (e.g., chloride, bromide, and iodide),methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl),trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonate. Apreferred leaving group is selected from nitrophenol;N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol;tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol;triflate; imidazole; dichlorophenol; tetrachlorophenol;1-hydroxybenzotriazole; tosylate; mesylate;2-ethyl-5-phenylisoxazolium-3′-sulfonate, anhydrides formed its self, orformed with the other anhydride, e.g. acetyl anhydride, formylanhydride; or an intermediate molecule generated with a condensationreagent for peptide coupling reactions or for Mitsunobu reactions.

The following abbreviations may be used herein and have the indicateddefinitions: Boc, tert-butoxy carbonyl; BroP,bromotrispyrrolidinophosphonium hexafluorophosphate; CDI,1,1′-carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCE,dichloroethane; DCM, dichloromethane; DEAD is diethylazodicarboxylate,DIAD, diisopropylazodicarboxylate; DIBAL-H, diisobutyl-aluminiumhydride; DIPEA or DEA, diisopropylethylamine; DEPC, diethylphosphorocyanidate; DMA, N,N-dimethyl acetamide; DMAP, 4-(N,N-dimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO,dimethylsulfoxide; DTPA is diethylenetriaminepentaacetic acid; DTT,dithiothreitol; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride; ESI-MS, electrospray mass spectrometry; EtOAc is ethylacetate; Fmoc is N-(9-fluorenylmethoxycarbonyl); HATU,O-(7-azabenzotriazol-1-yl)-N, N, N′, N′-tetramethyluroniumhexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high pressureliquid chromatography; NHS, N-Hydroxysuccinimide; MeCN is acetonitrile;MeOH is methanol; MMP, 4-methylmorpholine; PAB, p-aminobenzyl; PBS,phosphate-buffered saline (pH 7.0-7.5); Ph is phenyl; phe isL-phenylalanine; PyBrop is bromo-tris-pyrrolidino-phosphoniumhexafluorophosphate; PEG, polyethylene glycol; SEC, size-exclusionchromatography; TCEP, tris(2-carboxyethyl)phosphine; TFA,trifluoroacetic acid; THF, tetrahydrofuran; Val, valine; TLC is thinlayer chromatography; UV is ultraviolet.

The “amino acid(s)” can be natural and/or unnatural amino acids,preferably alpha-amino acids. Natural amino acids are those encoded bythe genetic code, which are alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tyrosine. tryptophan and valine. The unnatural amino acidsare derived forms of proteinogenic amino acids. Examples includehydroxyproline, lanthionine, 2-aminoisobutyric acid, dehydroalanine,gamma-aminobutyric acid (the neurotransmitter), ornithine, citrulline,beta alanine (3-aminopropanoic acid), gamma-carboxyglutamate,selenocysteine (present in many noneukaryotes as well as mosteukaryotes, but not coded directly by DNA), pyrrolysine (found only insome archaea and one bacterium), N-formylmethionine (which is often theinitial amino acid of proteins in bacteria, mitochondria, andchloroplasts), 5-hydroxytryptophan, L-dihydroxyphenylalanine,triiodothyronine, L-3,4-dihydroxyphenylalanine (DOPA), andO-phosphoserine. The term amino acid also includes amino acid analogsand mimetics. Analogs are compounds having the same general H₂N(R)CHCO₂Hstructure of a natural amino acid, except that the R group is not onefound among the natural amino acids. Examples of analogs includehomoserine, norleucine, methionine-sulfoxide, and methionine methylsulfonium. Preferably, an amino acid mimetic is a compound that has astructure different from the general chemical structure of analpha-amino acid but functions in a manner similar to one. The term“unnatural amino acid” is intended to represent the “D” stereochemicalform, the natural amino acids being of the “L” form. When 1˜8 aminoacids are used in this patent application, amino acid sequence is thenpreferably a cleavage recognition sequence for a protease. Many cleavagerecognition sequences are known in the art. See, e.g., Matayoshi et al.Science 247: 954 (1990); Dunn et al. Meth. Enzymol. 241: 254 (1994);Seidah et al. Meth. Enzymol. 244: 175 (1994); Thornberry, Meth. Enzymol.244: 615 (1994); Weber et al. Meth. Enzymol. 244: 595 (1994); Smith etal. Meth. Enzymol. 244: 412 (1994); and Bouvier et al. Meth. Enzymol.248: 614 (1995); the disclosures of which are incorporated herein byreference. In particular, the sequence is selected from the groupconsisting of Val-Cit, Ala-Val, Ala-Ala, Val-Val, Val-Ala-Val, Lys-Lys,Ala-Asn-Val, Val-Leu-Lys, Cit-Cit, Val-Lys, Ala-Ala-Asn, Asp-Lys,Asp-Glu, Glu-Lys, Lys, Cit, Ser, and Glu.

The “glycoside” is a molecule in which a sugar group is bonded throughits anomeric carbon to another group via a glycosidic bond. Glycosidescan be linked by an O-(an O-glycoside), N-(a glycosylamine), S-(athioglycoside), or C-(a C-glycoside) glycosidic bond. Its core theempirical formula is C_(m)(H₂O)_(n) (where m could be different from n,and m and n are <36), Glycoside herein includes glucose (dextrose),fructose (levulose) allose, altrose, mannose, gulose, iodose, galactose,talose, galactosamine, glucosamine, sialic acid, N-acetylglucosamine,sulfoquinovose (6-deoxy-6-sulfo-D-glucopyranose), ribose, arabinose,xylose, lyxose, sorbitol, mannitol, sucrose, lactose, maltose,trehalose, maltodextrins, raffinose, Glucuronic acid (glucuronide), andstachyose. It can be in D form or L form, 5 atoms cyclic furanose forms,6 atoms cyclic pyranose forms, or acyclic form, α-isomer (the —OH of theanomeric carbon below the plane of the carbon atoms of Haworthprojection), or a β-isomer (the —OH of the anomeric carbon above theplane of Haworth projection). It is used herein as a monosaccharide,disaccharide, polyols, or oligosaccharides containing 3-6 sugar units.

The term “antibody,” as used herein, refers to a full-lengthimmunoglobulin molecule or an immunologically active portion of afull-length immunoglobulin molecule, i.e., a molecule that contains anantigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce auto-immune antibodiesassociated with an autoimmune disease. The immunoglobulin disclosedherein can be of any type (e.g. IgG, IgE, IgM, IgD, IgA and IgY), class(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. The immunoglobulins can be derived from anyspecies. Preferably, however, the immunoglobulin is of human, murine, orrabbit origin.

Antibodies useful in the invention are preferably monoclonal, andinclude, but are not limited to, polyclonal, monoclonal, bispecific,human, humanized or chimeric antibodies, single chain antibodies, Fv,Fab fragments, F(ab′) fragments, F(ab′)₂ fragments, fragments producedby a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR's,and epitope-binding fragments of any of the above whichimmunospecifically bind to cancer cell antigens, viral antigens ormicrobial antigens.

An “enantiomer”, also known as an “optical isomer”, is one of twostereoisomers that are mirror images of each other that arenon-superposable (not identical), much as one's left and right hands arethe same except for being reversed along one axis (the hands cannot bemade to appear identical simply by reorientation). A single chiral atomor similar structural feature in a compound causes that compound to havetwo possible structures which are non-superposable, each a mirror imageof the other. The presence of multiple chiral features in a givencompound increases the number of geometric forms possible, though theremay be some perfect-mirror-image pairs. Enantiopure compounds refer tosamples having, within the limits of detection, molecules of only onechirality. When present in a symmetric environment, enantiomers haveidentical chemical and physical properties except for their ability torotate plane-polarized light (+/−) by equal amounts but in oppositedirections (although the polarized light can be considered an asymmetricmedium). They are sometimes called optical isomers for this reason. Amixture of equalparts of an optically active isomer and its enantiomeris termed racemic and has zero net rotation of plane-polarized lightbecause the positive rotation of each (+) form is exactly counteractedby the negative rotation of a (−) one. Enantiomer members often havedifferent chemical reactions with other enantiomer substances. Sincemany biological molecules are enantiomers, there is sometimes a markeddifference in the effects of two enantiomers on biological organisms. Indrugs, for example, often only one of a drug's enantiomers isresponsible for the desired physiologic effects, while the otherenantiomer is less active, inactive, or sometimes even productive ofadverse effects. Owing to this discovery, drugs composed of only oneenantiomer (“enantiopure”) can be developed to enhance thepharmacological efficacy and sometimes eliminate some side effects.

Isotopes are variants of a particular chemical element which differs inneutron number. All isotopes of a given element have the same number ofprotons in each atom. Each atomic number identifies a specific element,but not the isotope; an atom of a given element may have a wide range inits number of neutrons. The number of nucleons (both protons andneutrons) in the nucleus is the atom's mass number, and each isotope ofa given element has a different mass number. For example, carbon-12,carbon-13 and carbon-14 are three isotopes of the element carbon withmass numbers 12, 13 and 14 respectively. The atomic number of carbon is6, which means that every carbon atom has 6 protons, so that the neutronnumbers of these isotopes are 6, 7 and 8 respectively. Hydrogen atom hasthree isotopes of protium (¹H), deuterium (H), and tritium (H), whichdeuterium has twice the mass of protium and tritium has three times themass of protium. Isotopic substitution can be used to determine themechanism of a chemical reaction and via the kinetic isotope effect.Isotopic substitution can be used to study how the body affects aspecific xenobiotic/chemical after administration through the mechanismsof absorption and distribution, as well as the metabolic changes of thesubstance in the body (e.g. by metabolic enzymes such as cytochrome P450or glucuronosyltransferase enzymes), and the effects and routes ofexcretion of the metabolites of the drug. This study is calledpharmacokinetics (PK). Isotopic substitution can be used to study of thebiochemical and physiologic effects of drugs. The effects can includethose manifested within animals (including humans), microorganisms, orcombinations of organisms (for example, infection). This study is calledpharmacodynamics (PD). The effects can include those manifested withinanimals (including humans), microorganisms, or combinations of organisms(for example, infection). Both together influence dosing, benefit, andadverse effects of the drug. isotopes can contain a stable(non-radioactive) or an unstable element. Isotopic substitution of adrug may have a different thrapeutical efficacy of the original drug.

“Pharmaceutically” or “pharmaceutically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate.

“Pharmaceutically acceptable solvate” or “solvate” refer to anassociation of one or more solvent molecules and a disclosed compound.Examples of solvents that form pharmaceutically acceptable solvatesinclude, but are not limited to, water, isopropanol, ethanol, methanol,DMSO, ethyl acetate, acetic acid and ethanolamine.

“Pharmaceutically acceptable excipient” includes any carriers, diluents,adjuvants, or vehicles, such as preserving or antioxidant agents,fillers, disintegrating agents, wetting agents, emulsifying agents,suspending agents, solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions as suitabletherapeutic combinations.

As used herein, “pharmaceutical salts” refer to derivatives of thedisclosed compounds wherein the parent compound is modified by makingacid or base salts thereof. The pharmaceutically acceptable saltsinclude the conventional non-toxic salts or the quaternary ammoniumsalts of the parent compound formed, for example, from non-toxicinorganic or organic acids. For example, such conventional non-toxicsalts include those derived from inorganic acids such as hydrochloric,hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; andthe salts prepared from organic acids such as acetic, propionic,succinic, tartaric, citric, methanesulfonic, benzenesulfonic,glucuronic, glutamic, benzoic, salicylic, toluenesulfonic, oxalic,fumaric, maleic, lactic and the like. Further addition salts includeammonium salts such as tromethamine, meglumine, epolamine, etc., metalsalts such as sodium, potassium, calcium, zinc or magnesium.

The pharmaceutical salts of the present invention can be synthesizedfrom the parent compound which contains a basic or acidic moiety byconventional chemical methods. Generally, such salts can be prepared viareaction the free acidic or basic forms of these compounds with astoichiometric amount of the appropriate base or acid in water or in anorganic solvent, or in a mixture of the two. Generally, non-aqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, the disclosure of which is hereby incorporated byreference.

“Administering” or “administration” refers to any mode of transferring,delivering, introducing or transporting a pharmaceutical drug or otheragent to a subject. Such modes include oral administration, topicalcontact, intravenous, intraperitoneal, intramuscular, intralesional,intranasal, subcutaneous or intrathecal administration. Alsocontemplated by the present invention is utilization of a device orinstrument in administering an agent. Such device may utilize active orpassive transport and may be slow-release or fast-release deliverydevice.

In the context of cancer, the term “treating” includes any or all of:preventing growth of tumor cells or cancer cells, preventing replicationof tumor cells or cancer cells, lessening of overall tumor burden andameliorating one or more symptoms associated with the disease.

In the context of an autoimmune disease, the term “treating” includesany or all of: preventing replication of cells associated with anautoimmune disease state including, but not limited to, cells capable ofproducing an autoimmune antibody, lessening the autoimmune-antibodyburden and ameliorating one or more symptoms of an autoimmune disease.

In the context of an infectious disease, the term “treating” includesany or all of: preventing the growth, multiplication or replication ofthe pathogen that causes the infectious disease and ameliorating one ormore symptoms of an infectious disease.

Examples of a “mammal” or “animal” include, but are not limited to, ahuman, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat,bird and fowl.

The novel conjugates disclosed herein use the bridge linkers. Examplesof some suitable linkers and their synthesis are shown in FIGS. 1 to 34.

A CONJUGATE OF A CELL-BINDING AGENT-A CYTOTOXIC MOLECULE VIA THE SIDECHAIN-LINKAGE

In one aspect of the present invention, a conjugate containing a sidechain-linkage is represented by Formula (I):

wherein

“

” represents a single bond; n is 1 to 30;

T is a cell-binding agent/molecule, selected from the group consistingof an antibody, a single chain antibody, an antibody fragment that bindsto a target cell, a monoclonal antibody, a single chain monoclonalantibody, a monoclonal antibody fragment that binds to the target cell,a chimeric antibody, a chimeric antibody fragment that binds to thetarget cell, a domain antibody, a domain antibody fragment that binds tothe target cell, an adnectin that mimics antibody, DARPins, alymphokine, a hormone, a vitamin, a growth factor, a colony stimulatingfactor, a nutrient-transport molecule (a transferrin), and/or acell-binding peptide, protein, or small molecule attached on albumin, apolymer, a dendrimer, a liposome, a nanoparticle, a vesicle, or on a(viral) capsid;

L₁ and L₂ are a chain of atoms selected from C, N, O, S, Si, and P,preferably having 0˜500 atoms, which covalently connects to W and V₁,and V₁ and V₂. The atoms used in forming the Li and L₂ may be combinedin all chemically relevant ways, such as forming alkylene, alkenylene,and alkynylene, ethers, polyoxyalkylene, esters, amines, imines,polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides,carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids,peptides, acyloxylamines, hydroxamic acids, or combination abovethereof. Preferably L₁ and L₂ are, the same or different, independentlyselected from O, NH, N, S, P, NNH, NHNH, N(R₃), N(R₃)N(R₃′), CH, CO,C(O)NH, C(O)O, NHC(O)NH, NHC(O)O, polyethyleneoxy unit of formula(OCH₂CH₂)_(p)OR₃, or (OCH₂CH—(CH₃))_(p)OR₃, or NH(CH₂CH₂O)_(p)R₃, orNH(CH₂CH(CH₃)O)_(p)R₃, or N[(CH₂CH₂O)_(p)R₃]-[(CH₂CH₂O)_(p)′R₃′], or(OCH₂CH₂)_(p)COOR₃, or CH₂CH₂(OCH₂CH₂)_(p)COOR₃, wherein p and p′ areindependently an integer selected from 0 to about 1000, or combinationthereof, C₁-C₈ of alkyl; C₂-C₈ of heteroalkyl, alkylcycloalkyl,heterocycloalkyl; C₃-C₈ of aryl, Ar-alkyl, heterocyclic, carbocyclic,cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or(Aa)_(r), r=1-12(one to 12 amino acid units), which is composed fromnatural or unnatural amino acids, or the same or different sequences ofdipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide,heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide ordodecapeptide unit;

W is a stretcher unit having C₁-C₁₈, normally a self-immolative spacer,a peptidic unit, a hydrazone, a disulfide, a thioether, an ester, or anamide bond; w is 1 or 2 or 3;

V₁ and V₂ are independently a spacer unit and selected from O, NH, S,C₁-C₈ alkyl, C₂-C₈ heteroalkyl, alkenyl, or alkynyl, C₃-C₈ aryl,heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, oralkylcarbonyl, or (Aa)_(r), r=1-12(one to 12 amino acid units), which iscomposed from a natural or unnatural amino acid, or the same ordifferent sequences of dipeptide, tripeptide, tetrapeptide,pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide,decapeptide, undecapeptide or dodecapeptide unit; or (CH₂CH₂O)_(p), p is0-1000; and v₁ and v₂ are independently 0, 1 or 2, but v₁ and v₂ are 0at the same time; when v₁ or v₂ is 0, it means one of the side chain Q1or Q2 fragment is absent.

Q₁ and Q₂ are independently represented by Formula (I-q1):

wherein

is the site linked to L₁ or L₂; G₁ and G₂ are independently OC(O),NHC(O), C(O), CH₂, NH, OC(O)NH, NHC(O)NH, O, S, B, P(O)(OH), NHP(O)(OH),NHP(O)(OH)NH, CH₂P(O)(OH)NH, OP(O)(OH)O, CH₂P(O)(OH)O, NHS(O)₂,NHS(O)₂NH, CH₂S(O)₂NH, OS(O)₂O, CH₂S(O)₂O, Ar, ArCH₂, ArO, ArNH, ArS,ArNR₁, or (Aa)_(q1); G₃ is OH, SH, OR₁, SR₁, OC(O)R₁, NHC(O)R₁, C(O)R₁,CH₃, NH₂, NR₁, +NH(R₁), +N(R₁)(R₂), C(O)OH, C(O)NH₂, NHC(O)NH₂, BH₂,BR₁R₂, P(O)(OH)₂, NHP(O)(OH)₂, NHP(O)(NH₂)₂, S(O)₂(OH),(CH₂)_(q1)C(O)OH, (CH₂)_(q1)P(O)(OH)₂, C(O)(CH₂)_(q1)C(O)OH,OC(O)(CH₂)_(q1)C(O)OH, NHC(O)(CH₂)_(q1)C(O)OH, CO(CH₂)_(q1)P(O)(OH)₂,NHC(O)O(CH₂)_(q1)C(O)OH, OC(O)NH(CH₂)_(q1)C(O)OH,NHCO(CH₂)_(q1)P(O)(OH)₂, NHC(O)(NH)(CH₂)_(q1)C(O)OH,CONH(CH₂)_(q1)P(O)(OH)₂, NHS(O)₂(CH₂)_(q1)C(O)OH, CO(CH₂)_(q1)S(O)₂(OH),NHS(O)₂NH(CH₂)_(q1)C(O)OH, OS(O)₂NH(CH₂)_(q1)C(O)OH,NHCO(CH₂)_(q1)S(O)₂(OH), NHP(O)(OH)(NH)(CH₂)_(q1)C(O)OH,CONH(CH₂)_(q1)S(O)(OH), OP(O)(OH)₂, (CH₂)_(q1)P(O)(NH)₂, NHS(O)₂(OH),NHS(O)₂NH₂, CH₂S(O)₂NH₂, OS(O)₂OH, OS(O)₂OR₁, CH₂S(O)₂OR₁, Ar, ArR₁,ArOH, ArNH₂, ArSH, ArNHR₁, or (Aa)_(q1); (Aa)_(q1) is a peptidecontaining the same or different sequence of natural or unnatural aminoacids; X₁ and X₂ are independently O, CH₂, S, S(O), NHNH, NH, N(R₁),+NH(R₁), +N(R₁)(R₂), C(O), OC(O), OC(O)O, OC(O)NH, NHC(O)NH; Y₂ is O,NH, NR₁, CH₂. S, NHNH, Ar; p₁, p₂ and p₃ are independently 0-100 but arenot 0 at the same time; q₁ and q₂ are independently 0-24; R₁, R₂, R₃ andR₃′ are independently H, C₁-C₈ alkyl; C₂-C₈ heteroalkyl, orheterocyclic; C₃-C₈ aryl, Ar-alkyl, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroalkylcycloalkyl, carbocyclic, or alkylcarbonyl;

Preferably Q₁ and Q₂ are independently a C₂-C₁₀₀ polycarboxylacid, aC₂-C₉₀ polyalkylamine, a C₆-C₉₀ oligosaccharide or polysaccharide, aC₆-C₁₀₀ zwitterionic betaines or zwitterionic poly(sulfobetaine)) (PSB)sthat consist of a quaternary ammonium cation and a sulfonate anion, aC₆-C₁₀₀ biodegradable polymer, such as composed of poly (lactic/glycolicacid) (PLGA), poly(acrylates), chitosan, copolymer ofN-(2-hydroxypropyl)methacrylamide, poly[2-(methacryloyloxy)ethylphosphorylcholine] (PMPC), poly-L-glutamic acid,poly(lactide-co-glycolide) (PLG), poly(lactide-co-glycolide),Poly(ethylene glycol)(PEG), poly(propylene glycol)(PPG),poly(lactide-co-glycolide), poly(ethylene glycol)-modified peptides,poly(ethylene glycol)-containing an aminoacid or peptides, poly(ethyleneglycol)-modified lipids, poly(ethylene glycol)-modified alkylcarboxicacid, poly(ethylene glycol)-modified alkylamine,poly(lactide-co-glycolide, hyaluronic acid (HA) (glycosaminoglycan),heparin/heparan sulfate (HSGAGs), chondroitin sulfate/dermatan sulfate(CSGAGs), poly(ethylene glycol)-modified alkylsulfate, poly(ethyleneglycol)-modified alkylphosphate, or poly(ethylene glycol)-modified alkylquaternary ammonium;

Example Structures of Q₁ and Q₂ are Shown Below

wherein R₂₅ and R₂₅′ are independently selected from H; HC(O), CH₃C(O),CH₃C(NH), C₁-C₁₈ alkyl, C₁-C₁₈ alkyl, alkyl-Y₁-SO₃H, C₁-C₁₈alkyl-Y₁-PO₃H₂, C₁-C₁₈ alkyl-Y₁-CO₂H, C₁-C₁₈ alkyl-Y₁-N⁺R₁′R₂′R₃′R⁴′,C₁-C₁₈ alkyl-Y₁-CONH₂, C₂-C₁₈ alkylene, C₂-C₁₈ ester, C₂-C₁₈ ether,C₂-C₁₈ amine, C₂-C₁₈ alkyl carboxylamide, C₃-C₁₈ Aryl, C₃-C₁₈ cyclicalkyl, C₃-C₁₈ heterocyclic, 1-24 amino acids; C₂-C₁₈ lipid, a C₂-C₁₈fatty acid or a C₂-C₁₈ fatty ammonium lipid; X₁ and X₂ are independentlyselected from NH, N(R₁′), O, CH₂, S, C(O), S(O), S(O₂), P(O)(OH), NHNH,CH═CH, Ar or (Aa)_(q1), q₁=0-24 (0-24 amino acids, q₁=0 means absent);X₁, X₂, X₃, X₄, Y₁, Y₂ and Y₃ are independently selected from NH,N(R₁′), O, C(O), CH₂, S, S(O), NHNH, C(O), OC(O), OC(O)O, OC(O)NH,NHC(O)NH, Ar or Ar or (Aa)_(q1), X₁, X₂, X₃, X₄, Y₁, Y₂ and Y₃ can beindependently absent; p₁, p₂ and p₃ are independently 0-100 but are not0 at the same time; q₁, q₂ and q₃ are independently 0-24; R₁′, R₂′, R₃′and R⁴′ are independently selected from H and C₁-C₆ alkyl; Aa is naturalor unnatural amino acid; Ar or (Aa)_(q1), is the same or differentsequence of peptides; q₁=0 means (Aa)_(q1) absent;

D is tubulysin analog having the following formula (II):

or a pharmaceutically acceptable salt, hydrates, or hydrated salt; or apolymorphic crystalline structure; or an optical isomer, racemate,diastereomer or enantiomer thereof,

wherein

is a linkage site that links to W independently;

wherein R¹, R₂, R³, and R⁴ are independently H, C₁˜C₈ alkyl; C₂˜C₈heteroalkyl, or heterocyclic; C₃˜C₈ aryl, Ar-alkyl, cycloalkyl,alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbocyclic,or alkylcarbonyl; or R¹R², R′R³, R²R³, R³R⁴, R⁵R⁶, R¹¹R¹² or R¹³R¹⁴ forma 3˜7 membered carbocyclic, cycloalkyl, heterocyclic, heterocycloalkyl,aromatic or heteroaromatic ring system; R¹ and R² can be independentlyabsent when they link to W independently or simultaneously, Y¹ is N orCH;

wherein R⁵, R⁶, R⁸, R¹⁰ and R¹¹ are independently H, or C₁˜C₄ alkyl orheteroalkyl;

wherein R⁷ is independently H, R¹⁴, —R¹⁴C(═O)X¹R¹⁵; or —R¹⁴X¹R¹⁵; X¹ isO, S, S—S, NH, CH₂ or NR¹⁴;

wherein R⁹ is selected from H, OH, —O—, ═O, —OR¹⁴, —OC(═O)R¹⁴,—OC(═O)NHR¹⁴—, —OC(═O) R¹⁴SSR¹⁵—, OP(═O)(OR¹⁴)—, —OC(═O)NR¹⁴R¹⁵,OP(═O)(OR¹⁴), or OR¹⁴OP(═O)(OR¹⁵);

wherein R¹¹ is independently H, R¹⁴, —R¹⁴C(═O)R¹⁶, —R¹⁴X²R¹⁶,—R¹⁴C(═O)X², wherein X² is —O—, —S—, —NH—, —N(R¹⁴)—, —O—R¹⁴—, —S—R¹⁴—,—S(═O)—R¹⁴—, or —NHR¹⁴; wherein R¹² is R¹⁵,—OH, —SH, —NH₂, NH, NHNH₂,—NH(R¹⁵), —OR¹⁵, —R¹⁵COR¹⁶, —R¹⁵COOR¹⁶, —R¹⁵C(O)NH₂, —R¹⁵C(O)NHR¹⁷,—SR¹⁶, R¹⁵S(═O)R¹⁶, — R¹⁵P(═O)(OR¹⁷)₂, —R¹⁵OP(═O)(OR¹⁷)₂,—CH₂OP(═O)(OR¹⁷)₂, —R¹⁵SO₂R¹⁷, —R¹⁵X²R¹⁶, —R¹⁵C(═O)X², where X² is —O—,OH, SH, —S—, NH₂, —NH—, —N(R¹⁵)—, —O—R¹⁵—, —S—R¹⁵—, —S(═O)—R¹⁵—, CH₂or—NHR¹⁵—;

R¹³ and R¹⁴ are independently H, O, S, NH, N(R¹⁵), NHNH, —OH, —SH, —NH₂,NH, NHNH₂, —NH(R¹⁵), —OR¹⁵, CO, —COX², —COX²R¹⁶, R¹⁷, F, Cl, Br, I,SR¹⁶, NR¹⁶R¹⁷, N═NR¹⁶, N═R¹⁶, NO₂, SOR¹⁶R¹⁷, SO₂R¹⁶, SO₃R¹⁶, PR¹⁶R¹⁷,POR¹⁶R¹⁷, PO₂R¹⁶R¹⁷, OP(O)(OR¹⁷)₂, OCH₂OP(O)(OR¹⁷)₂, OC(O)R¹⁷,OC(O)OP(O)(OR¹⁷)₂, PO(OR¹⁶)(OR¹⁷), OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂, OC(O)NHR¹⁷;—O—(C₄-C₁₂ glycoside), -N-(C₄-C₁₂ glycoside); C₁˜C₈ alkyl, heteroalkyl;C₂-C₈ of alkenyl, alkynyl, heteroalkyl, heterocycloalkyl; C₃-C₈ of aryl,Ar-alkyl, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl,heteroaryl, or 2-8 carbon atoms of esters, ether, or amide; or peptidescontaining 1-8 amino acids (NH(Aa)_(1˜8) or CO(Aa)_(1˜8) (N-terminal orC-terminal 1-8 the same or different amino acids), or polyethyleneoxyunit of formula (OCH₂CH₂)_(p) or (OCH₂CH(CH₃))_(p), wherein p is aninteger from 0 to about 1000, or combination of above groups thereof; X²is O, S, S—S, NH, CH₂, OH, SH, NH₂, CHR¹⁴ or NR¹⁴;

R⁵, R¹⁶ and R¹⁷ is independently H, C₁˜C₈ alkyl, heteroalkyl; C₂˜C₈ ofalkenyl, alkynyl, heteroalkyl, heterocycloalkyl; C₃-C₈ of aryl,Ar-alkyl, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl,heteroaryl, alkylcarbonyl, or Na⁺, K⁺, Cs⁺, Li⁺, Ca²⁺, Mg⁺, Zn²⁺,N⁺(R¹)(R²)(R³) (R⁴), HN⁺(C₂H₅OH)₃ salt;

Y¹ and Y² are independently N or CH; q is 0 or 1; when q=0, Y³ does notexist, Y⁴, Y⁵, Y⁶ and Y⁷ are independently CH, N, NH, O, S, or N (R₁),thus Y², Y⁴ Y⁵ Y⁶ and Y⁷form a heteroaromatic ring of furan, pyrrolethiophene, thiazole, oxazole and imidazole, pyrazole, triazole,tetrazole, thiadiazole; when q=1, Y³, Y⁴, Y⁵, Y⁶ and Y⁷ areindependently CH or N, thus Y², Y³, Y⁴, Y⁵, Y⁶ and Y⁷ form aromatic ringof benzene, pyridine, pyridazine, pyrimidine, pyrazine, triazine,tetrazine, pentazine;

Examples of the Tubulysin Structures are Shown Below

wherein R²⁰ is H; C₁-C₈ of linear or branched alkyl or heteroalkyl;C₂-C₈ of linear or branched alkenyl, alkynyl, alkylcycloalkyl,heterocycloalkyl; C₃-C₈ linear or branched of aryl, Ar-alkyl,heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl,alkylcarbonyl, heteroaryl; carbonate (—C(O)OR¹⁷), carbamate(—C(O)NR¹⁷R¹⁸); or 1-8 carbon atoms of carboxylate, esters, ether, oramide; or 1-8 amino acids; or polyethyleneoxy unit of formula(OCH₂CH₂)_(p) or (OCH₂CH(CH₃))_(p), wherein p is an integer from 0 toabout 1000; or R²⁰ is absent and the oxygen forms a ketone, orcombination above thereof, Z³ and Z³ are independently H, O H, N H₂, O,NH, COOH, COO, C(O),C(O), C(O)NH, C(O)NH₂, R¹⁸, OCH₂OP(O)(OR¹⁸)₂,OC(O)OP(O)(OR¹⁸)₂, OPO(OR¹⁸)₂, NHPO(OR¹⁸)₂, OP(O)(OR¹⁸)OP(O)(OR¹⁸)₂,OC(O)R¹⁸, OC(O)NHR¹⁸, OSO₂(OR¹⁸), O—(C₄-C₁₂-glycoside), of linear orbranched alkyl or heteroalkyl; C₂-C₈ of linear or branched alkenyl,alkynyl, alkylcycloalkyl, heterocycloalkyl; C₃-C₈ linear or branched ofaryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl,heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; carbonate (—C(O)OR¹⁷),carbamate (—C(O)NR¹⁷R¹⁸); R¹⁷ and R¹⁸ are independently H, linear orbranched alkyl or heteroalkyl; C₂-C₈ of linear or branched alkenyl,alkynyl, alkylcycloalkyl, heterocycloalkyl; C₃-C₈ linear or branched ofaryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl,heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; carbonate (—C(O)OR¹⁷),carbamate (—C(O)NR¹⁷R¹⁸); R¹⁹ is H, OH, NH₂, OSO₂(OR¹⁸),XCH₂OP(O)(OR¹⁸)₂, XPO(OR¹⁸)₂, XC(O)OP(O)(OR¹⁸)₂, XC(O)R¹⁸, XC(O)NHR¹⁸,C₁˜C₈ alkyl or carboxylate; C₂˜C₈ alkenyl, alkynyl, alkylcycloalkyl,heterocycloalkyl; C₃˜C₈ aryl or alkylcarbonyl; or pharmaceutical salts;X is O, S, NH, NHNH, or CH₂; R⁷ is defined the same above.

Additionally W, L₁, L₂, V₁, and V₂, may independently be composed of oneor more linker components of 6-maleimidocaproyl (“MC”),maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”),alanine-phenylalanine (“ala-phe” or “af”), p-aminobenzyloxy-carbonyl(“PAB”), 4-thiopentanoate (“SPP”), 4—(N-maleimidomethyl)cyclohexane-1carboxylate (“MCC”), (4-acetyl)amino-benzoate (“SIAB”), 4-thio-butyrate(SPDB), 4-thio-2-hydroxysulfonyl-butyrate (2-Sulfo-SPDB), as thestructures shown below or natural or unnatural peptides having 1˜12natural or unnatural amino acid unites. The natural aminoacid ispreferably selected from aspartic acid, glutamic acid, arginine,histidine, lysine, serine, threonine,

natural or unnatural peptides containing 1-20 amino acids;

W, L₁, L₂ V₁, and V₂ may also independently contain a self-immolative ora non-self-immolative component, peptidic units, a hydrazone bond, adisulfide, an ester, an oxime, an amide, or a thioether bond. Theself-immolative unit includes, but is not limited to, aromatic compoundsthat are electronically similar to the para-aminobenzylcarbamoyl (PAB)groups such as 2-aminoimidazol-5-methanol derivatives, heterocyclic PABanalogs, beta-glucuronide, and ortho or para-aminobenzylacetals;

Preferably, the self-immolative linker component has one of thefollowing structures:

wherein the (*) atom is the point of attachment of additional spacer orreleasable linker units, or the cytotoxic agent, and/or the bindingmolecule (CBA); X¹, Y¹, Z² and Z³ are independently NH, O, or S; Z¹ isindependently H, NHR₁, OR₁, SR₁, COX₁R₁, wherein X₁ and R₁ are definedabove; v is 0 or 1; U¹ is independently H, OH, C₁˜C₆ alkyl,(OCH₂CH₂)_(n), F, Cl, Br, I, OR₅, SR₅, NR₅R₅′, N═NR₅, N═R, NR₅R₅′, NO₂,SOR₅R₅′, SO₂R₅, SO₃R₅, SO₃R₅, PR₅R₅′, POR₅R₅′, PO₂R₅R₅′, OPO(OR₅)(OR₅′),or OCH₂PO(OR₅(OR₅′), wherein R₅ and R₅′ are independently selected fromH, C₁˜C₈ of alkyl; C₂˜C₈ of alkenyl, alkynyl, heteroalkyl, or aminoacid; C₃-C₈ of aryl, heterocyclic, carbocyclic, cycloalkyl,heterocycloalkyl, heteroaralkyl, alkylcarbonyl, or glycoside; orpharmaceutical cation salts;

W, L₁, L₂ V₁, and V₂ may also independently contain non-self-immolativelinker component having one of the following structures:

wherein the (*) atom is the point of attachment of additional spacer orreleasable linkers, the cytotoxic agents, and/or the binding molecules;X¹, Y¹, U¹, R₅, R₅′ are defined as above; r is 0˜100; m and n are 0˜6independently;

Further preferably, W, L₁, L₂ V₁, and V₂ may independently be areleasable linker component. The term releasable refers to a linker thatincludes at least one bond that can be broken under physiologicalconditions, such as a pH-labile, acid-labile, base-labile, oxidativelylabile, metabolically labile, biochemically labile or enzyme-labilebond. It is appreciated that such physiological conditions resulting inbond breaking do not necessarily include a biological or metabolicprocess, and instead may include a standard chemical reaction, such as ahydrolysis or substitution reaction, for example, an endosome having alower pH than cytosolic pH, and/or disulfide bond exchange reaction witha intracellular thiol, such as a millimolar range of abundant ofglutathione inside the malignant cells;

Examples of the releasable components of W, L₁, L₂ V₁, and V₂independently include, but not limited:

—(CR₅R₆)_(m)(Aa)_(r)(CR₇R₈)_(n)(OCH₂CH₂)_(t)—,—(CR₅R₆)_(m)(CR₇R₈)_(n)(Aa)_(r)(OCH₂CH₂)_(t)—,—(Aa)_(r)—(CR₅R₆)_(m)(CR₇R₈)_(n)(OCH₂CH₂)_(t)—,—(CR₅R₆)_(m)(CR₇R₈)_(n)(OCH₂CH₂)_(r)(Aa)_(t)—,—(CR₅R₆)_(m)—(CR₇═CR₈)(CR₉R₁₀)_(n)(Aa)_(t)(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)(NR₁₁CO)(Aa)_(t)(CR₉R₁₀)_(n)—(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)(Aa)_(t)(NR₁₁CO)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)(OCO)(Aa)_(t)(CR₉R₁₀)_(n)—(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)(OCNR₇)(Aa)_(t)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)(CO)(Aa)_(t)—(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)(NR₁₁CO)(Aa)_(t)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)—(OCO)(Aa)_(t)(CR₉R₁₀)_(n)—(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)(OCNR₇)(Aa)_(t)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)(CO)(Aa)_(t)(CR₉R₁₀)_(n)—(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)-phenyl-CO(Aa)_(t)(CR₅R₆)_(n)—,—(CR₅R₆)_(m)—furyl-CO(Aa)_(t)(CR₇R₈)_(n)—,—(CR₅R₆)_(m)—oxazolyl-CO(Aa)_(t)(CR₇R₈)_(n)—,—(CR₅R₆)_(m)-thiazolyl-CO(Aa)_(t)(CCR₇R₈)_(n)—,—(CR₅R₆)_(t)-thienyl-CO(CR₇R₈)_(n)—,—(CR₅R₆)_(t)-imidazolyl-CO—(CR₇R₈)_(n)—,—(CR₅R₆)_(t)-morpholino-CO(Aa)_(t)—(CR₇R₈)_(n)—,—(CR₅R₆)_(t)piperazino-CO(Aa)_(t)—(CR₇R₈)_(n)—,—(CR₅R₆)_(t)-N-methylpiperazin-CO(Aa)_(t)—(CR₇R₈)_(n)—,—(CR₇R₈)_(m)-(Aa)_(t)phenyl-, —(CR₅R₆)_(m)-(Aa)_(t)furyl-,—(CR₅R₆)_(m)-oxazolyl(Aa)_(t)—, —(CR₅R₆)_(m)—thiazolyl(Aa)_(t)—,—(CR₅R₆)_(m)-thienyl-(Aa)_(t)—, —(CR₅R₆)_(m)-imidazolyl(Aa)_(t)—,—(CR₅R₆)_(m)-morpho-lino-(Aa)_(t)—, —(CR₅R₆)_(m)-piperazino-(Aa)_(t)—,—(CR₅R₆)_(m)-N-methylpiperazino-(Aa)_(t)—,-K(CR₅R₆)_(m)—(Aa)_(r)(CR₇R₈)_(n)(OCH₂CH₂)_(t)—,—K(CR₅R₆)_(m)(CR₇R₈)_(n)(Aa)_(r)(OCH₂CH₂)_(t)—,—K(Aa)_(r)—(CR₅R₆)_(m)—(CR₇R₈)_(n)(OCH₂CH₂)_(t)—,—K(CR₅R₆)_(m)(CR₇R₈)_(n)(OCH₂CH₂)_(r)(Aa)_(t)—,—K(CR₅R₆)_(m)—(CR₇═CR₅)—(CR₉R₁₀)_(n)(Aa)_(t)(OCH₂CH₂)_(r)—,—K(CR₅R₆)_(m)(NR₁₁CO)(Aa)_(t)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)-,—K(CR₅R₆)_(m)(Aa)_(t)(NR₁₁CO)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₅R₆)_(m)(OCO)(Aa)_(t)(CR₉R₁₀)_(n)—(OCH₂CH₂)_(r)—,—K(CR₅R₆)_(m)(OCNR₇)(Aa)_(t)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₅R₆)_(m)(CO)(Aa)_(t)—(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₅R₆)_(m)(NR₁₁CO)(Aa)_(t)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₅R₆)_(m)—(OCO)(Aa)_(t)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₅R₆)_(m)(OCNR₇)(Aa)_(t)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—K—(CR₅R₆)_(m)(CO)(Aa)_(t)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₅R₆)_(m)-phenyl-CO(Aa)_(t)(CR₇R₈)_(n)—,—K—(CR₅R₆)_(m)—furyl-CO(Aa)_(t)—(CR₇R₈)_(n)—,—K(CR₅R₆)_(m)-oxazolyl-CO(Aa)_(t)(CR₇R₈)_(n)—,—K(CR₅R₆)_(m)—thiazolyl-CO(Aa)_(t)—(CR₇R₈)_(n)—,—K(CR₅R₆)_(t)-thienyl-CO(CR₇R₈)_(n)—,—K(CR₅R₆)_(t)imidazolyl-CO—(CR₇R₈)_(n)—,—K(CR₅R₆)_(t)morpholino-CO(Aa)_(t)(CR₇R₈)_(n)—,—K(CR₅R₆)_(t)piperazino-CO(Aa)_(t)—(CR₇R₈)_(n)—,—K(CR₅R₆)_(t)-N-methylpiperazinCO(Aa)_(t)(CR₇R₈)_(n)—,—K(CR₅R)_(m)(Aa)_(t)phenyl, —K—(CR₅R₆)_(m)-(Aa)_(t)furyl-,—K(CR₅R₆)_(m)-oxazolyl(Aa)_(t)—, —K(CR₅R₆)_(m)-thiazolyl(Aa)_(t)—,—K(CR₅R₆)_(m)—thienyl-(Aa)_(t)—, —K(CR₅R₆)_(m)-imidazolyl(Aa)_(t)—,—K(CR₅R₆)_(m)-morpholino(Aa)_(t)—, —K(CR₅Rb)_(m)—piperazino-(Aa)_(t)G,—K(CR₅R₆)_(m)N-methylpiperazino(Aa)_(t)—; wherein m, Aa, m, n, R₃, R₄,and R₅ are described above; t and r here are 0-100 independently; R₆,R₇, R₅, R₉, and R₁₀ are independently chosen from H; halide; C₁˜C₈ ofalkyl, aryl, alkenyl, alkynyl, ether, ester, amine or amide, whichoptionally substituted by one or more halide, CN, NR₁R₂, CF₃, OR₁, Aryl,heterocycle, S(O)R₁, SO₂R₁, —CO₂H, —SO₃H, —OR₁, —CO₂R₁, —CONR₁,—PO₂R₁R₂, —PO₃H or P(O)R₁R₂R₃; K is NR₁, —SS—, —C(═O)—, —C(═O)NH—,—C(═O)O—, —C═NH—O—, —C═O)—, —N—NH—, —C(═O)NH—NH—, O, S, Se, B, Het(heterocyclic or heteroaromatic ring having C₃-C₈), or peptidescontaining 1-20 amino acids;

Additionally components of W, L₁, L₂ V₁, and V₂ may independentlycontain one of the following hydrophilic structures:

wherein

is the site of linkage; X₂, X₃, X₄, X₅, or X₆, are independentlyselected from NH; NHNH; N(R₃); N(R₃)N(R₃′); O; S; C₁-C₆ of alkyl; C₂-C₆of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C₃˜C₈ of aryl,Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl,alkylcarbonyl, heteroaryl; or 1-8 amino acids; Wherein R₃ and R₃′ areindependently H;C₁-C₈ of alkyl; C₂-C₈ of hetero-alkyl, alkylcycloalkyl,heterocycloalkyl; C₃-C₈ of aryl, Ar-alkyl, heterocyclic, carbocyclic,cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or 1-8carbon atoms of esters, ether, or amide; or polyethyleneoxy unit offormula (OCH₂CH₂)_(p) or (OCH₂CH(CH₃))_(p), wherein p is an integer from0 to about 1000, or combination above thereof;

More preferably, components of W, L₁, L₂ V₁, and V₂ are independentlylinear alkyl having from 1-6 carbon atoms, or polyethyleneoxy unit offormula (OCH₂CH₂)_(p), p=1˜5000, or a peptide containing 1˜4 units ofaminoacids (L or D form), or combination above.

Alternatively, any one or more of W, Q₁, Q₂, L₁, L₂, V₁, or V₂, can beindependently absent but Q₁, and Q₂ are not absent at the same time.

Generally stated, in another aspect, when V₁ and/or V₂ linked to thecell-binding molecule, T, or when L1 and/or L2 directly linked to T(wherein V₁, and V₂, are absent), it could have one or more of thefollowing structures of the linkage:

wherein R²⁰ and R²¹ are independently C₁˜C₈ alkyl; C₂˜C₈ heteroalkyl, orheterocyclic; C₃˜C₈ aryl, Ar-alkyl, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroalkylcycloalkyl, carbocyclic, or alkylcarbonyl;or C₂-C₁₀₀ polyethylene glycol having formula of (CH₂CH₂O)_(p), p isdefined above.

In another further aspect, Q₁ and Q₂ are preferably selected from apolyalkylene glycol containing a C₂-C₁₈ lipid, or a C₂-C₁₈ fatty acid,or a C₂-C₁₈ fatty ammonium lipid. The polyalkylene glycol chain not onlyhelps the conjugate more hydrophilic during the production, but alsoprevents the conjugate linker from hydrolysis by a hydrolase, e.g. aproteinase or an esterase. The lipid can help the conjugate to bind toan albumin in mammal bloods and then leads to the conjugate slowlydissociation from this complex during the blood circulation. Thus, theside chain linker of the present patent application makes the conjugatemore stable in the circulation. Poly alkylene glycols here include, butare not limited to, poly(ethylene glycols) (PEGs), poly(propyleneglycol) and copolymers of ethylene oxide and propylene oxide;particularly preferred are PEGs, and more particularly preferred aremonofunctionally activated hydroxyPEGs (e.g., hydroxyl PEGs activated ata single terminus, including reactive esters ofhydroxyPEG-monocarboxylic acids, hydroxyPEG-monoaldehydes,hydroxyPEG-monoamines, hydroxyPEG-monohydrazides,hydroxyPEG-monocarbazates, hydroxyl PEG-monoiodoacetamides, hydroxylPEG-monomaleimides, hydroxyl PEG-monoorthopyridyl disulfides,hydroxyPEG-monooximes, hydroxyPEG-monophenyl carbonates, hydroxylPEG-monophenyl glyoxals, hydroxyl PEG-monothiazolidine-2-thiones,hydroxyl PEG-monothioesters, hydroxyl PEG-monothiols, hydroxylPEG-monotriazines and hydroxyl PEG-monovinylsulfones). The polyalkyleneglycol has a molecular weight of from about 10 Daltons to about 200 kDa,preferably about 88 Da to about 40 kDa; two branch chains each with amolecular weight of about 88 Da to about 40 kDa; and more preferably twobranches, each of about 88 Da to about 20 kDa. In one particularembodiment, the polyalkylene glycol is poly(ethylene) glycol and has amolecular weight of about 10 kDa; about 20 kDa, or about 40 kDa. Inspecific embodiments, the PEG is a PEG 10 kDa (linear or branched), aPEG 20 kDa (linear or branched), or a PEG 40 kDa (linear or branched). Anumber of US patents have disclosed the preparation of linear orbranched “non-antigenic” PEG polymers and derivatives or conjugatesthereof, e.g., U.S. Pat. Nos. 5,428,128; 5,621,039; 5,622,986;5,643,575; 5,728,560; 5,730,990; 5,738,846; 5,811,076; 5,824,701;5,840,900; 5,880,131; 5,900,402; 5,902,588; 5,919,455; 5,951,974;5,965,119; 5,965,566; 5,969,040; 5,981,709; 6,011,042; 6,042,822;6,113,906; 6,127,355; 6,132,713; 6,177,087, and 6,180,095.

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers; wherein Z³ andZ³ are independently H, OH, NH₂, O, NH, COOH, COO, C(O),C(O), C(O)NH,C(O)NH₂, R¹⁸, OCH₂OP(O)(OR¹⁸)₂, OC(O)OP(O)(OR¹⁸)₂, OPO(OR¹⁸)₂,NHPO(OR¹⁸)₂, OP(O)(OR¹⁸)OP(O)(OR¹⁸)₂, OC(O)R¹⁸, OC(O)NHR¹⁸, OSO₂(OR¹⁸),O-(C₄-C₁₂-glycoside), of linear or branched alkyl or heteroalkyl; C₂-C₈of linear or branched alkenyl, alkynyl, alkylcycloalkyl,heterocycloalkyl; C₃-C₈ linear or branched of aryl, Ar-alkyl,heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl,alkylcarbonyl, heteroaryl; carbonate (—C(O)OR¹⁷), carbamate(—C(O)NR¹⁷R¹⁸); or polyalkylene glycols have a molecular weight of fromabout 88 Daltons to about 20 kDa; R¹⁷ and R¹⁸ are independently H,linear or branched alkyl or heteroalkyl; C₂-C₈ of linear or branchedalkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C₃-C₈ linear orbranched of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl,heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; carbonate (—C(O)OR¹⁷),carbamate (—C(O)NR¹⁷R¹⁸); R¹⁹ is H, OH, NH₂, OSO₂(OR¹⁸),XCH₂OP(O)(OR¹⁸)₂, XPO(OR¹⁸)₂, XC(O)OP(O)(OR¹⁸)₂, XC(O)R¹⁸, XC(O)NHR¹⁸,C₁˜C₈ alkyl or carboxylate; C₂˜C₈ alkenyl, alkynyl, alkylcycloalkyl,heterocycloalkyl; C₃˜C₈ aryl or alkylcarbonyl; or pharmaceutical salts;X, X₁, X₂ and X₃ are independently O, S, NH, NHNH, or CH₂; q₁, q₂ and q₃are independently selected from 0-24; p, p₁ and p₂ are independently1-100; R₁′, R₂′, R₃′ and R⁴′ are independently selected from H and C₁-C₆alkyl; Aa is natural or unnatural amino acid; r is 0-12; (Aa)_(r) is apeptide containing the same or different sequence of amino acids whenr>2; r=0 means (Aa)_(r) absent; m and n are independently 1-30.

In another aspect of the present invention, a conjugate containing aside chain-linkage is represented by Formula (III):

wherein D, W, w, L₁, L₂, Q₁, Q₂, V₁, V₂, v₁, v₂, n, T are defined thesame as in Formula (I).

Examples of Formula (III) Structures are as Following

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers; wherein X₁,X₂, X₃, m, n, (Aa)_(r), p₁, p₂, p₃, q₁, q₂ are described above.

In another aspect of the present invention, the side chain-linkagecompound is represented by Formula (IV), which can readily react to acell-binding molecule T, or to a modified cell-binding molecule T toform a conjugate of Formula (I):

wherein D, W, w, L₁, L₂, Q₁, Q₂, V₁, V₂, v₁, v₂, and n, are defined thesame as in Formula (I);

L_(V1) is a reacting group that can be reacted with a thiol, amine,carboxylic acid, selenol, phenol or hydroxyl group on a cell-bindingmolecule. Such reacting groups are, but are not limited to, a halide(e.g., fluoride, chloride, bromide, and iodide), methanesulfonyl(mesyl), toluenesulfonyl (tosyl), trifluoromethyl-sulfonyl (triflate),trifluoromethylsulfonate, nitrophenoxyl, N-succinimidyloxyl (NHS),phenoxyl; dinitrophenoxyl; pentafluorophenoxyl, tetrafluorophenoxyl,trifluorophenoxyl, difluorophenoxyl, monofluorophenoxyl,pentachloro-phenoxyl, 1H-imidazole-1-yl, chlorophenoxyl,dichlorophenoxyl, trichlorophenoxyl, tetrachlorophenoxyl,N-(benzotriazol-yl)oxyl, 2-ethyl-5-phenylisoxazolium-3′-sulfonyl,phenyloxadiazole-sulfonyl (-sulfone-ODA),2-ethyl-5-phenylisoxazolium-yl, phenyloxadiazol-yl (ODA), oxadiazol-yl,unsaturated carbon (a double or a triple bond between carbon-carbon,carbon-nitrogen, carbon-sulfur, carbon-phosphrus, sulfur-nitrogen,phosphrus-nitrogen, oxygen-nitrogen, or carbon-oxygen), or anintermediate molecule generated with a condensation reagent forMitsunobu reactions. The examples of condensation reagents are: EDC(N-(3-Dimethyl-aminopropyl)-N′-ethylcarbodiimide), DCC(Dicyclohexyl-carbodiimide), N,N′-Diisopropyl-carbodiimide (DIC),N-Cyclohexyl-N′-(2-morpholino-ethyl)carbodiimidemetho-p-toluenesulfonate (CMC,or CME-CDI), 1,1′-Carbonyldiimi-dazole(CDI), TBTU (O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate),N,N,N′,N′-Tetramethyl-O—(1H-benzotriazol-1-yl)-uroniumhexafluorophosphate (HBTU),(Benzotriazol-1-yloxy)tris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP),(Benzotriazol-1-yloxy)tripyrroli-dinophosphonium hexafluorophosphate(PyBOP), Diethyl cyanophosphonate (DEPC),Chloro-N,N,N′,N′-tetramethylformamidiniumhexafluorophosphate,1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophos-phate (HATU),1-[(Dimethylami-no)(morpholino)methylene]-1H-[1,2,3]triazolo[4,5-b]pyridine-1-ium3-oxide hexafluoro-phosphate (HDMA),2-Chloro-1,3-dimethyl-imidazolidinium hexafluorophosphate (CIP),Chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP),Fluoro-N,N,N′,N′-bis(tetramethylene)formamidinium hexafluorophosphate(BTFFH), N,N,N′,N′-Tetramethyl-S-(1-oxido-2-pyridyl)thiuroniumhexafluorophosphate, O-(2-Oxo-1(2H)pyridyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TPTU),S-(1-Oxido-2-pyridyl)-N,N,N′,N′-tetramethylthiuronium tetrafluoroborate,O-[(Ethoxycarbonyl)-cyanomethylenamino]-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HOTU),(1-Cyano-2-ethoxy-2-oxoethylidenamino-oxy)dimethylamino-morpholino-carbeniumhexafluorophosphate (COMU),O-(Benzotriazol-1-yl)-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate (HBPyU), N-Benzyl-N′-cyclohexyl-carbodiimide (with,or without polymer-bound), Dipyrrolidino(N-succinimidyl-oxy)carbeniumhexafluoro-phosphate (HSPyU), Chlorodipyrrolidinocarbeniumhexafluoro-phosphate (PyClU), 2-Chloro-1,3-dimethylimidazolidiniumtetrafluoroborate(CIB), (Benzotriazol-1-yloxy)dipiperidino-carbeniumhexafluorophosphate (HBPipU),O-(6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TCTU), Bromotris(dimethylamino)-phosphoniumhexafluorophosphate (BroP), Propylphosphonic anhydride (PPACA, T3P®),2-Morpholinoethyl isocyanide (MEI),N,N,N′,N′-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate(HSTU), 2-Bromo-1-ethyl-pyridinium tetrafluoro-borate (BEP),0-[(Ethoxycarbonyl)cyano-methylenamino]-N,N,N′,N′-tetra-methyluroniumtetrafluoroborate (TOTU),4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholiniumchloride (MMTM,DMTMM), N,N,N′,N′-Tetramethyl-O-(N-succinimidyl)uroniumtetrafluoroborate (TSTU),O-(3,4-Dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoro-borate (TDBTU),1,1′-(Azodicarbonyl)-dipiperidine (ADD),Di-(4-chlorobenzyl)-azodicarboxylate (DCAD), Di-tert-butylazodicarboxylate (DBAD),Diisopropyl azodicarboxylate (DIAD), Diethylazodicarboxylate (DEAD). In addition, L_(V1) and L_(V2) can be ananhydride, formed by acid themselves or formed with other C₁-C₈ acidanhydrides; Preferably L_(V1) is selected from, a halide (e.g.,fluoride, chloride, bromide, and iodide), methanesulfonyl (mesyl),toluenesulfonyl (tosyl), trifluoromethyl-sulfonyl (triflate),trifluoromethylsulfonate, nitrophenoxyl, N-succinimidyloxyl (NHS),phenoxyl; dinitrophenoxyl; pentafluorophenoxyl, tetrafluorophenoxyl,trifluorophenoxyl, difluorophenoxyl, monofluoro-phenoxyl,pentachlorophenoxyl, 1H-imidazole-1-yl, chlorophenoxyl,dichlorophenoxyl, trichlorophenoxyl, tetrachlorophenoxyl,N-(benzotriazol-yl)oxyl, 2-ethyl-5-phenylisoxazolium-3′-sulfonyl,phenyloxadiazole-sulfonyl (-sulfone-ODA),2-ethyl-5-phenylisoxazolium-yl, phenyloxadiazol-yl (ODA), oxadiazol-yl,unsaturated carbon (a double or a triple bond between carbon-carbon,carbon-nitrogen, carbon-sulfur, carbon-phosphrus, sulfur-nitrogen,phosphrus-nitrogen, oxygen-nitrogen, or carbon-oxygen), or one of thefollowing structure:

wherein X₁′ is F, Cl, Br, I or L_(v3); X₂′ is O, NH, N(R₁), or CH₂; R₃is independently H, aromatic, heteroaromatic, or aromatic group whereinone or several H atoms are replaced independently by —R₁, -halogen,—OR₁, —SR₁, -NR₁R₂, — NO₂, —S(O)R₁,—S(O)₂R₁, or —COOR₁; L_(v3) is aleaving group selected from F, Cl, Br, I, nitrophenol;N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol;tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol;triflate; imidazole; dichlorophenol; tetrachlorophenol;1-hydroxybenzotriazole; tosylate; mesylate;2-ethyl-5-phenylisoxazolium-3′-sulfonate, anhydrides formed its self, orformed with the other anhydride, e.g. acetyl anhydride, formylanhydride; or an intermediate molecule generated with a condensationreagent for peptide coupling reactions or for Mitsunobu reactions.

Examples of Formula (IV) are Shown Below

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers; wherein X₁,X₂, X₃, Z₂, Z₃, p, p₁, p₂, p₃, q₁, q₂, L_(v3), (Aa)_(r), R₂₅, R₂₅′, andm are described above.In another aspect of the present invention, the side chain-linkagecompound is represented by Formula (V), which can readily react to acell-binding molecule T to form a conjugate of Formula (III):

wherein D, W, w, L₁, L₂, Q₁, Q₂, V₁, V₂, v₁, v₂, and n, are defined thesame as in Formula (I); wherein L_(V1) and L_(V2) have independently thesame definition of L_(V1) in formula (IV) and both L_(V1) and L_(V2) canbe the same or different in Formula (V).

Examples of Formula (V) are Shown Below

wherein X₁, X₂, X₃, Z₂, Z₃, p. p₁, p₂, p₃, q₁, q₂ L_(v3), (Aa)_(r), R₂R₂and m are described above.

The present invention further relates to a method of making acell-binding molecule-tubulysin analog conjugate of Formula (I) andFormula (III) as well the application of the conjugates of Formula (I)and Formula (I).

A cell-binding agent/molecule, T, can be any kind presently known, orthat become known, of a molecule that binds to, complexes with, orreacts with a moiety of a cell population sought to be therapeuticallyor otherwise biologically modified. Preferably the cell-bindingagent/molecule is an immunotherapeutic protein, an antibody, a singlechain antibody; an antibody fragment that binds to the target cell; amonoclonal antibody; a single chain monoclonal antibody; or a monoclonalantibody fragment that binds the target cell; a chimeric antibody; achimeric antibody fragment that binds to the target cell; a domainantibody; a domain antibody fragment that binds to the target cell;adnectins that mimic antibodies; DARPins; a lymphokine; a hormone; avitamin; a growth factor; a colony stimulating factor; or anutrient-transport molecule (a transferrin); a binding peptides havingover four aminoacids, or protein, or antibody, or small cell-bindingmolecule or ligand attached on albumin, polymers, dendrimers, liposomes,nanoparticles, vesicles, or (viral) capsids;

Preferably L_(V1) and L_(V2) react to pairs of thiols of a cell-bindingagent/molecule. The thiols are preferably pairs of sulfur atoms reducedfrom the inter chain disulfide bonds of the cell-binding agent by areducing agent selected from dithiothreitol (DTT), dithioerythritol(DTE), L-glutathione (GSH), tris (2-carboxyethyl) phosphine (TCEP),2-mercaptoethylamine (β-MEA), or/and beta mercaptoethanol (j-ME, 2-ME);

THE PREPARATION OF THE CONJUGATES OF A TUBULYSIN ANALOG TO A CELLBINDING MOLECULES VIA A SIDE CHAIN-LINKAGE

The preparation of the conjugates of a tubulysin analog to a cellbinding molecules of the present invention and the synthetic routes toproduce the conjugates via side chain-linkage are shown in FIGS. 1-51 .

The conjugates of Formula (I) and (III) can be prepared through theintermediate compounds of Formula (IV) and (V) respectively. In general,tubulysin analogs of Formula (IV) and (V) are synthesized to have thefunction groups of Lv1 and Lv2 that can be readily reacted to acell-binding molecule or a modified cell-binding molecule. The synthesisof tubulysin analogs of Formula (IV) and (V) and some preparations ofFormula (I) and (III) are structurally shown in the FIGS. 1-51 .

To synthesize the conjugate of Formula (I), in general, a function groupL_(V1) on Formula (IV) reacts one, two or more residues of a cellbinding molecule at 0-60° C., pH 5-9 aqueous media with or withoutaddition of 0˜30% of water mixable (miscible) organic solvents, such asDMA, DMF, ethanol, methanol, acetone, acetonitrile, THF, isopropanol,dioxane, propylene glycol, or ethylene diol, following by dialysis orchromatographic purification to form a conjugate compound of Formula(I). Some of the residue (reacting group for conjugation) of thecell-binding molecule can be obtained through protein engineering.

The conjugates of the Formula (III) can also be obtained through thereaction of the function group L_(V1) and L_(V2) of linkers of theFormula (V) to two or more residues of a cell binding molecule,preferably a pair of free thiols generated through reduction ofdisulfide bonds of the cell-binding molecule at 0-60° C., pH 5˜9 aqueousmedia with or without addition of 0˜30% of water mixable (miscible)organic solvents, to form the conjugate molecule. The pairs of thiolsare preferred pairs of disulfide bonds reduced from the inter chaindisulfide bonds of the cell-binding agent by a reducing agent which canselected from dithiothreitol (DTT), dithioerythritol (DTE),L-glutathione (GSH), tris (2-carboxyethyl) phosphine (TCEP),2-mercaptoethylamine (β-MEA), or/and beta mercaptoethanol (β-ME, 2-ME)at pH4˜9 aqueous media with or without addition of 0˜30% of watermixable (miscible) organic solvents.

The reactive groups of L_(V1) and L_(V2) on Formula (IV) and Formula(V), which can be independently disulfide, thiol, thioester, maleimido,halogen substituted maleimidoes, haloacetyl, azide, 1-yne, ketone,aldehyde, alkoxyamino, triflate, carbonylimidazole, tosylate, mesylate,2-ethyl-5-phenylisoxazolium-3′-sulfonate, or carboxyl acid esters ofnitrophenol, N-hydroxysuccinimide (NHS), phenol; dinitrophenol,pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol,pentachlorophenol, dichlorophenol, tetrachlorophenol,1-hydroxybenzotriazole, anhydrides, or hydrazide groups, or other acidester derivatives, can react to one, two or more groups on acell-binding molecule/agent, simultaneously or sequentially at 0-60° C.,pH 4-9.5 aqueous media with or without addition of 0˜30% of watermixable (miscible) organic solvents, to yield a conjugate of the Formula(I) and Formula (III), after column purification or dialysis. Thereactive groups of L_(V1) and L_(V2) on Formula (IV) and Formula (V)react to the modified cell-binding molecule in different waysaccordingly. For example, a linkage containing disulfide bonds in acell-binding agent-tubulysin analog conjugate of Formula (I) is achievedby a disulfide exchange between the disulfide bond in the modifiedcell-binding agent and L_(V1) and L_(V2) having a free thiol group, orby a disulfide exchange between a free thiol group in the modifiedcell-binding agent and a disulfide bond on L_(V1) and/or L_(V2). Inorder to swift the disulfide exchange reaction, the disulfide groupnormally are a group of disulfanylpyridine, disulfanyl-nitropyridine,disulfanyl-nitrobenzene, disulfanyl-nitrobenzoic acid, ordisulfanyl-dinitrobenzene, etc. A linkage containing thioether bonds inthe conjugates of Formula (I) and Formula (III) is achieved by reactionof the maleimido or haloacetyl or ethylsulfonyl either on a modifiedcell-binding agent or a tubulysin analog of Formula (IV) and Formula (V)to a free thiol group on a tubulysin analog of Formula (IV) and Formula(V) or on a modified cell-binding agent respectively; A linkagecontaining a bond of an acid labile hydrazone in the conjugates can beachieved by reaction of a carbonyl group of the drug of Formula (IV) andFormula (V) or of cell-binding molecule with the hydrazide moiety on amodified cell-binding molecule or on the drug of Formula (IV) andFormula (V) accordingly, by methods known in the art (see, for example,P. Hamann et al., Cancer Res. 53, 3336-34, 1993; B. Laguzza et al., J.Med. Chem., 32; 548-55, 1959; P. Trail et al., Cancer Res., 57; 100-5,1997); A linkage containing a bond of triazole in the conjugates can beachieved by reaction of a 1-yne group of the drug of Formula (IV) andFormula (V) or of cell-binding molecule with the azido moiety on theother counterpart accordingly, through the click chemistry (Huisgencycloaddition) (Lutz, J-F. et al, 2008, Adv. Drug Del. Rev.60, 958-70;Sletten, E. M. et al 2011, AccChem. Research 44, 666-76). A linkagecontaining a bond of oxime in the conjugates linked via oxime isachieved by reaction of a group of a ketone or aldehyde group of thedrug of Formula (IV) and Formula (V) or of a cell-binding molecule witha group of oxylamine on the other counterpart respectively. Athiol-containing cell-binding molecule can react with the drug moleculelinker of Formula (IV) and Formula (V) bearing a maleimido, or ahaloacetyl, or an ethylsulfonyl substituent at pH 5.5˜9.0 in aqueousbuffer to give a thioether linkage conjugate of Formula (I) and Formula(III). A thiol-containing cell-binding molecule can undergo disulfideexchange with a drug linker of Formula (IV) and Formula (V) bearing apyridyldithio moiety to give a conjugate having a disulfide bondlinkage. A cell-binding molecule bearing a hydroxyl group or a thiolgroup can be reacted with a drug linker of Formula (IV) and Formula (V)bearing a halogen, particularly the alpha halide of carboxylates, in thepresence of a mild base, e.g. pH 8.0˜9.5, to give a modified drugbearing an ether or thiol ether linkage. A hydroxyl or an amino group ona cell-binding molecule can be condensed with a cross drug linker ofFormula (IV) and Formula (V) bearing a carboxyl group, in the presenceof a dehydrating agent, such as EDC or DCC, to give ester linkage. Acell-binding molecule containing an amino group can condensate with agroup of carboxyl ester of NHS, imidazole, nitrophenol;N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol;tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol;triflate; imidazole;dichlorophenol;tetrachlorophenol;1-hydroxyben-zotriazole; tosylate;mesylate; or 2-ethyl-5-phenylisoxazolium-3′-sulfonate on the drug-linkerof Formula (IV) and Formula (V) to give a conjugate via amide bondlinkage.

The synthetic conjugate may be purified by standard biochemical means,such as gel filtration on a Sephadex G25 or Sephacryl S300 column,adsorption chromatography, and ion exchange or by dialysis. In somecases, a small molecule as a cell-binding agent (e.g. folic acid,melanocyte stimulating hormone, EGF etc.) conjugated with a smallmolecular drugs can be purified by chromatography such as by HPLC,medium pressure column chromatography or ion exchange chromatography.

In order to achieve a higher yield of conjugation reaction for theFormula (I) or Formula (III) with a pair of free thiols on thecell-binding molecule, preferably on an antibody, a small percentage ofwater miscible organic solvents, or phase transfer agents, may berequired to add to the reaction mixture. To cross-linking reagent(linker) of Formula (IV) or Formula (V) can be first dissolved in apolar organic solvent that is miscible with water, for example indifferent alcohols, such as methanol, ethanol, and propanol, acetone,acetonitrile, tetrahydrofuran (THF), 1,4-dioxane, dimethyl formamide(DMF), dimethyl acetamide (DMA), or dimethylsulfoxide (DMSO) at a highconcentration, for example 1˜500 mM. Meanwhile, the cell-bindingmolecule, such as antibody dissolved in an aqueous buffer pH 4˜9.5,preferably pH 6˜8.5, at 1˜50 mg/ml concentration was treated with 0.5˜20equivalent of TCEP or DTT for 20 min to 48 hour. After the reduction,DTT can be removed by SEC chromatographic purification. TCEP can beoptionally removed by SEC chromatography too, or staying in the reactionmixture for the next step reaction without further purification, butpreferably TCEP is neutralized with azide compounds, such as4-azidobenzoic acid, 4-(azidomethyl)benzoic acid, or azido-polyethyleneglycolyl (e.g. 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanol).Furthermore, the reduction of antibodies or the other cell-bindingagents with TCEP can be performed along with existing a drug-linkermolecule of Formula (IV) or Formula (V), for which the cross-linkingconjugation of the cell-binding molecules can be achieved simultaneouslyalong with the TCEP reduction.

The aqueous solutions for the modification of cell-binding agents arebuffered between pH 4 and 9, preferably between 6.0 and 7.5 and cancontain any non-nucleophilic buffer salts useful for these pH ranges.Typical buffers include phosphate, acetate, triethanolamine HCl, HEPES,and MOPS buffers, which can contain additional components, such ascyclodextrins, hydroxypropyl-O-cyclodextrin, polyethylene glycols,sucrose and salts, for examples, NaCl and KCl. After the addition of thedrug-linker of Formula (IV) or Formula (V) into the solution containingthe reduced cell-binding molecules, the reaction mixture is incubated ata temperature of from 4° C. to 45° C., preferably at 15° C.—ambienttemperature. The progress of the reaction can be monitored by measuringthe decrease in the absorption at a certain UV wavelength, such as at252 nm, or increase in the absorption at a certain UV wavelength, suchas 280 nm, or the other appropriate wavelength. After the reaction iscomplete, isolation of the modified cell-binding agent can be performedin a routine way, using for example a gel filtration chromatography, anion exchange chromatography, an adsorptive chromatography or columnchromatography over silica gel or alumina, crystallization, preparatorythin layer chromatography, ion exchange chromatography, or HPLC.

The extent of modification can be assessed by measuring the absorbanceof the nitropyridine thione, dinitropyridine dithione, pyridine thione,carboxylamidopyridine dithione and dicarboxyl-amidopyridine dithionegroup released via UV spectra. For the conjugation without a chromophoregroup, the modification or conjugation reaction can be monitored byLC-MS, preferably by HPLC-MS/MS, UPLC-QTOF mass spectrometry, orCapilary electrophoresis-mass spectrometry (CE-MS). The side chaincross-linkers described herein have diverse functional groups that canreact with any cell-binding molecules, particularly a modifiedcell-binding molecule that possess a suitable substituent. For examples,the modified cell-binding molecules bearing an amino or hydroxylsubstituent can react with drugs bearing an N-hydroxysuccinimide (NHS)ester, the modified cell-binding molecules bearing a thiol substituentcan react with drugs bearing a maleimido or haloacetyl group.Additionally, the modified cell-binding molecules bearing a carbonyl(ketone or aldehyde) substituent either through protein engineering,enzymatical reaction or chemical modification can react with drugsbearing a hydrazide or an alkoxyamine. One skilled in the art canreadily determine which modified drug-linker to be used based on theknown reactivity of the available functional group on the modifiedcell-binding molecules.

Cell-Binding Agents

The cell-binding molecule, Cb, that comprises the conjugates and themodified cell-binding agents of the present invention may be of any kindpresently known, or that become known, molecule that binds to, complexeswith, or reacts with a moiety of a cell population sought to betherapeutically or otherwise biologically modified.

The cell binding molecules/agents include, but are not limited to, largemolecular weight proteins such as, for example, antibody, anantibody-like protein, full-length antibodies (polyclonal antibodies,monoclonal antibodies, dimers, multimers, multispecific antibodies(e.g., bispecific antibodies); single chain antibodies; fragments ofantibodies such as Fab, Fab′, F(ab′)₂, Fv, [Parham, J. Immunol. 131,2895-902 (1983)], fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, CDR's, diabody, triabody,tetrabody, miniantibody, small immune proteins (SIP), andepitope-binding fragments of any of the above which immuno-specificallybind to cancer cell antigens, viral antigens, microbial antigens or aprotein generated by the immune system that is capable of recognizing,binding to a specific antigen or exhibiting the desired biologicalactivity (Miller et al (2003) J. of Immunology 170: 4854-61);interferons (such as type I, II, III); peptides; lymphokines such asIL-2, IL-3, IL-4, IL-5, IL-6, IL-10, GM-CSF, interferon-gamma (IFN-γ);hormones such as insulin, TRH (thyrotropin releasing hormones), MSH(melanocyte-stimulating hormone), steroid hormones, such as androgensand estrogens, melanocyte-stimulating hormone (MSH); growth factors andcolony-stimulating factors such as epidermal growth factors (EGF),granulocyte-macrophage colony-stimulating factor (GM-CSF), transforminggrowth factors (TGF), such as TGFα, TGFβ, insulin and insulin likegrowth factors (IGF-I, IGF-II) G-CSF, M-CSF and GM-CSF [Burgess,Immunology Today, 5, 155-8 (1984)]; vaccinia growth factors (VGF);fibroblast growth factors (FGFs); smaller molecular weight proteins,poly-peptide, peptides and peptide hormones, such as bombesin, gastrin,gastrin-releasing peptide; platelet-derived growth factors; interleukinand cytokines, such as interleukin-2 (IL-2), interleukin-6 (IL-6),leukemia inhibitory factors, granulocyte-macrophage colony-stimulatingfactor (GM-CSF); vitamins, such as folate; apoproteins andglycoproteins, such as transferrin [O'Keefe et al, 260 J. Biol. Chem.932-7 (1985)]; sugar-binding proteins or lipoproteins, such as lectins;cell nutrient-transport molecules; and small molecular inhibitors, suchas prostate-specific membrane antigen (PSMA) inhibitors and smallmolecular tyrosine kinase inhibitors (TKI), non-peptides or any othercell binding molecule or substance, such as bioactive polymers (Dhar, etal, Proc. Natl. Acad. Sci. 2008, 105, 17356-61); fusion proteins; kinaseinhibitors; gene-targeting agents; bioactive dendrimers (Lee, et al,Nat. Biotechnol. 2005, 23, 1517-26; Almutairi, et al; Proc. Natl. Acad.Sci. 2009, 106, 685-90); nanoparticles (Liong, et al, ACS Nano, 2008, 2,1309-12; Medarova, et al, Nat. Med. 2007, 13, 372-7; Javier, et al,Bioconjugate Chem. 2008, 19, 1309-12); liposomes (Medinai, et al, Curr.Phar. Des. 2004, 10, 2981-9); viral capsides (Flenniken, et al, VirusesNanotechnol. 2009, 327, 71-93).

In general, a monoclonal antibody is preferred as a cell-surface bindingagent if an appropriate one is available. And the antibody may bemurine, human, humanized, chimeric, or derived from other species.

Production of antibodies used in the present invention involves in vivoor in vitro procedures or combinations thereof. Methods for producingpolyclonal anti-receptor peptide antibodies are well-known in the art,such as in U.S. Pat. No. 4,493,795 (to Nestor et al). A monoclonalantibody is typically made by fusing myeloma cells with the spleen cellsfrom a mouse that has been immunized with the desired antigen (Kohler,G.; Milstein, C. (1975). Nature 256: 495-7). The detailed procedures aredescribed in “Antibodies--A Laboratory Manual”, Harlow and Lane, eds.,Cold Spring Harbor Laboratory Press, New York (1988), which isincorporated herein by reference. Particularly monoclonal antibodies areproduced by immunizing mice, rats, hamsters or any other mammal with theantigen of interest such as the intact target cell, antigens isolatedfrom the target cell, whole virus, attenuated whole virus, and viralproteins. Splenocytes are typically fused with myeloma cells usingpolyethylene glycol (PEG) 6000. Fused hybrids are selected by theirsensitivity to HAT (hypoxanthine-aminopterin-thymine). Hybridomasproducing a monoclonal antibody useful in practicing this invention areidentified by their ability to immunoreacted specified receptors orinhibit receptor activity on target cells.

A monoclonal antibody used in the present invention can be produced byinitiating a monoclonal hybridoma culture comprising a nutrient mediumcontaining a hybridoma that secretes antibody molecules of theappropriate antigen specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well-known techniques, such as using protein-A affinitychromatography; anion, cation, hydrophobic, or size exclusivechromatographies (particularly by affinity for the specific antigenafter protein A, and sizing column chromatography); centrifugation,differential solubility, or by any other standard technique for thepurification of proteins.

Media useful for the preparation of these compositions are bothwell-known in the art and commercially available and include syntheticculture media. An exemplary synthetic medium is Dulbecco's minimalessential medium (DMEM; Dulbecco et al., Virol. 8, 396 (1959))supplemented with 4.5 gm/l glucose, 0-20 mM glutamine, 0˜20% fetal calfserum, several ppm amount of heavy metals, such as Cu, Mn, Fe, or Zn,etc, or/and the other heavy metals added in their salt forms, and withan anti-foaming agent, such as polyoxyethylene-polyoxypropylene blockcopolymer.

In addition, antibody-producing cell lines can also be created bytechniques other than fusion, such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with an oncovirus, suchas Epstein-Barr virus (EBV, also called human herpesvirus 4 (HHV-4)) orKaposi's sarcoma-associated herpesvirus (KSHV). See, U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;4,472,500; 4,491,632; 4,493,890. A monoclonal antibody may also beproduced via an anti-receptor peptide or peptides containing thecarboxyl terminal as described well-known in the art. See Niman et al.,Proc. Natl. Acad. Sci. USA, 80: 4949-53 (1983); Geysen et al., Proc.Natl. Acad. Sci. USA, 82: 178-82 (1985); Lei et al. Biochemistry 34(20):6675-88, (1995). Typically, the anti-receptor peptide or a peptideanalog is used either alone or conjugated to an immunogenic carrier, asthe immunogen for producing anti-receptor peptide monoclonal antibodies.

There are also a number of other well-known techniques for makingmonoclonal antibodies as binding molecules in this invention.Particularly useful are methods of making fully human antibodies. Onemethod is phage display technology which can be used to select a rangeof human antibodies binding specifically to the antigen using methods ofaffinity enrichment. Phage display has been thoroughly described in theliterature and the construction and screening of phage display librariesare well known in the art, see, e.g., Dente et al, Gene. 148(1):7-13(1994); Little et al, Biotechnol Adv. 12(3): 539-55 (1994); Clackson etal., Nature 352: 264-8 (1991); Huse et al., Science 246: 1275-81 (1989).

Monoclonal antibodies derived by hybridoma technique from anotherspecies than human, such as mouse, can be humanized to avoid humananti-mouse antibodies when infused into humans. Among the more commonmethods of humanization of antibodies are complementarity-determiningregion grafting and resurfacing. These methods have been extensivelydescribed, see e.g. U.S. Pat. Nos. 5,859,205 and 6,797,492; Liu et al,Immunol Rev. 222: 9-27 (2008); Almagro et al, Front Biosci. 13: 1619-33(2008); Lazar et al, Mol Immunol. 44(8): 1986-98 (2007); L₁ et al, Proc.Natl. Acad. Sci. USA. 103(10): 3557-62 (2006) each incorporated hereinby reference.

Fully human antibodies can also be prepared by immunizing transgenicmice, rabbits, monkeys, or other mammals, carrying large portions of thehuman immunoglobulin heavy and light chains, with an immunogen. Examplesof such mice are: the Xenomouse. (Abgenix/Amgen), the HuMAb-Mouse(Medarex/BMS), the VelociMouse (Regeneron), see also U.S. Pat. Nos.6,596,541, 6,207,418, 6,150,584, 6,111,166, 6,075,181, 5,922,545,5,661,016, 5,545,806, 5,436,149 and 5,569,825. In human therapy, murinevariable regions and human constant regions can also be fused toconstruct called “chimeric antibodies” that are considerably lessimmunogenic in man than murine mAbs (Kipriyanov et al, Mol Biotechnol.26: 39-60 (2004); Houdebine, Curr Opin Biotechnol. 13: 625-9 (2002) eachincorporated herein by reference). In addition, site-directedmutagenesis in the variable region of an antibody can result in anantibody with higher affinity and specificity for its antigen (Branniganet al, Nat Rev Mol Cell Biol. 3: 964-70, (2002)); Adams et al, J ImmunolMethods. 231: 249-60 (1999)) and exchanging constant regions of a mAbcan improve its ability to mediate effector functions of binding andcytotoxicity.

Antibodies immunospecific for a malignant cell antigen can also beobtained commercially or produced by any method known to one of skill inthe art such as, e.g., chemical synthesis or recombinant expressiontechniques. The nucleotide sequence encoding antibodies immune-specificfor a malignant cell antigen can be obtained commercially, e.g., fromthe GenBank database or a database like it, the literature publications,or by routine cloning and sequencing.

Apart from an antibody, a peptide or protein that bind/block/target orin some other way interact with the epitopes or corresponding receptorson a targeted cell can be used as a binding molecule. These peptides orproteins could be any random peptide or proteins that have an affinityfor the epitopes or corresponding receptors and they don't necessarilyhave to be of the immune-globulin family. These peptides can be isolatedby similar techniques as for phage display antibodies (Szardenings, JRecept Signal Transduct Res. 2003, 23(4): 307-49). The use of peptidesfrom such random peptide libraries can be similar to antibodies andantibody fragments. The binding molecules of peptides or proteins may beconjugated on or linked to a large molecules or materials, such as, butis not limited, an albumin, a polymer, a liposome, a nano particle, adendrimer, as long as such attachment permits the peptide or protein toretain its antigen binding specificity.

Examples of antibodies used for conjugation of drugs via the linkers ofthis prevention for treating cancer, autoimmune disease, and/orinfectious disease include, but are not limited to, 3F8 (anti-GD2),Abagovomab (anti CA-125), Abciximab (anti CD41 (integrin alpha-IIb),Adalimumab (anti-TNF-α), Adecatumumab (anti-EpCAM, CD326), Afelimomab(anti-TNF-α); Afutuzumab (anti-CD20), Alacizumab pegol (anti-VEGFR2),ALD518 (anti-IL-6), Alemtuzumab (Campath, MabCampath, anti-CD52),Altumomab (anti-CEA), Anatumomab (anti-TAG-72), Anrukinzumab (IMA-638,anti-IL-13), Apolizumab (anti-HLA-DR), Arcitumomab (anti-CEA),Aselizumab (anti-L-selectin (CD62L), Atlizumab (tocilizumab, Actemra,RoActemra, anti-IL-6 receptor), Atorolimumab (anti-Rhesus factor),Bapineuzumab (anti-beta amyloid), Basiliximab (Simulect, antiCD25 (achain of IL-2 receptor), Bavituximab (anti-phosphatidylserine),Bectumomab (LymphoScan, anti-CD22), Belimumab (Benlysta, LymphoStat-B,anti-BAFF), Benralizumab (anti-CD125), Bertilimumab (anti-CCL11(eotaxin-1)), Besilesomab (Scintimun, anti-CEA-related antigen),Bevacizumab (Avastin, anti-VEGF-A), Biciromab (FibriScint, anti-fibrinII beta chain), Bivatuzumab (anti-CD44 v6), Blinatumomab (BiTE,anti-CD19), Brentuximab (cAC10, anti-CD30 TNFRSF8), Briakinumab(anti-IL-12, IL-23) Canakinumab (Ilaris, anti-IL-1), Cantuzumab (C242,anti-CanAg), Capromab, Catumaxomab (Removab, anti-EpCAM, anti-CD3), CC49(anti-TAG-72), Cedelizumab (anti-CD4), Certolizumab pegol (Cimziaanti-TNF-α), Cetuximab (Erbitux, IMC-C225, anti-EGFR), Citatuzumabbogatox (anti-EpCAM), Cixutumumab (anti-IGF-1), Clenoliximab (anti-CD4),Clivatuzumab (anti-MUC1), Conatumumab (anti-TRAIL-R2), CR6261(anti-Influenza A hemagglutinin), Dacetuzumab (anti-CD40), Daclizumab(Zenapax, anti-CD25 (a chain of IL-2 receptor)), Daratumumab (anti-CD38(cyclic ADP ribose hydrolase), Denosumab (Prolia, anti-RANKL), Detumomab(anti-B-lymphoma cell), Dorlimomab, Dorlixizumab, Ecromeximab (anti-GD3ganglioside), Eculizumab (Soliris, anti-C5), Edobacomab(anti-endotoxin), Edrecolomab (Panorex, MAb17-1A, anti-EpCAM),Efalizumab (Raptiva, anti-LFA-1 (CD11a), Efungumab (Mycograb,anti-Hsp90), Elotuzumab (anti-SLAMF7), Elsilimomab (anti-IL-6),Enlimomab pegol (anti-ICAM-1 (CD54)), Epitumomab (anti-episialin),Epratuzumab (anti-CD22), Erlizumab (anti-ITGB2 (CD18)), Ertumaxomab(Rexomun, anti-HER2/neu, CD3), Etaracizumab (Abegrin, anti-integrinα_(v)β₃), Exbivirumab (anti-hepatitis B surface antigen), Fanolesomab(NeutroSpec, anti-CD15), Faralimomab (anti-interferon receptor),Farletuzumab (anti-folate receptor 1), Felvizumab (anti-respiratorysyncytial virus), Fezakinumab (anti-IL-22), Figitumumab (anti-IGF-1receptor), Fontolizumab (anti-IFN-γ), Foravirumab (anti-rabies virusglycoprotein), Fresolimumab (anti-TGF-β), Galiximab (anti-CD80),Gantenerumab (anti-beta amyloid), Gavilimomab (anti-CD147 (basigin)),Gemtuzumab (anti-CD33), Girentuximab (anti-carbonic anhydrase 9),Glembatumumab (CR011, anti-GPNMB), Golimumab (Simponi, anti-TNF-α),Gomiliximab (anti-CD23 (IgE receptor)), Ibalizumab (anti-CD4),Ibritumomab (anti-CD20), Igovomab (Indimacis-125, anti-CA-125),Imciromab (Myoscint, anti-cardiac myosin), Infliximab (Remicade,anti-TNF-α), Intetumumab (anti-CD51), Inolimomab (anti-CD25 (a chain ofIL-2 receptor)), Inotuzumab (anti-CD22), Ipilimumab (anti-CD152),Iratumumab (anti-CD30 (TNFRSF8)), Keliximab (anti-CD4), Labetuzumab(CEA-Cide, anti-CEA), Lebrikizumab (anti-IL-13), Lemalesomab(anti-NCA-90 (granulocyte antigen)), Lerdelimumab (anti-TGF beta 2),Lexatumumab (anti-TRAIL-R2), Libivirumab (anti-hepatitis B surfaceantigen), Lintuzumab (anti-CD33), Lucatumumab (anti-CD40), Lumiliximab(anti-CD23 (IgE receptor), Mapatumumab (anti-TRAIL-R1), Maslimomab(anti-T-cell receptor), Matuzumab (anti-EGFR), Mepolizumab (Bosatria,anti-IL-5), Metelimumab (anti-TGF beta 1), Milatuzumab (anti-CD74),Minretumomab (anti-TAG-72), Mitumomab (BEC-2, anti-GD3 ganglioside),Morolimumab (anti-Rhesus factor), Motavizumab (Numax, anti-respiratorysyncytial virus), Muromonab-CD3 (Orthoclone OKT3, anti-CD3), Nacolomab(anti-C242), Naptumomab (anti-5T4), Natalizumab (Tysabri, anti-integrinα₄), Nebacumab (anti-endotoxin), Necitumumab (anti-EGFR), Nerelimomab(anti-TNF-α), Nimotuzumab (Theracim, Theraloc, anti-EGFR), Nofetumomab,Ocrelizumab (anti-CD20), Odulimomab (Afolimomab, anti-LFA-1 (CD11a)),Ofatumumab (Arzerra, anti-CD20), Olaratumab (anti-PDGF-R α), Omalizumab(Xolair, anti-IgE Fc region), Oportuzumab (anti-EpCAM), Oregovomab(OvaRex, anti-CA-125), Otelixizumab (anti-CD3), Pagibaximab(anti-lipoteichoic acid), Palivizumab (Synagis, Abbosynagis,anti-respiratory syncytial virus), Panitumumab (Vectibix,ABX-EGF,anti-EGFR), Panobacumab (anti-Pseudomonas aeruginosa),Pascolizumab (anti-IL-4), Pemtumomab (Theragyn, anti-MUC1), Pertuzumab(Omnitarg, 2C4,anti-HER2/neu), Pexelizumab (anti-C5), Pintumomab(anti-adenocarcinoma antigen), Priliximab (anti-CD4), Pritumumab(anti-vimentin), PRO 140 (anti-CCR5), Racotumomab (1E10,anti-(N-glycolylneuraminic acid (NeuGc, NGNA)-gangliosides GM3)),Rafivirumab (anti-rabies virus glycoprotein), Ramucirumab (anti-VEGFR2),Ranibizumab (Lucentis, anti-VEGF-A), Raxibacumab (anti-anthrax toxin,protective antigen), Regavirumab (anti-cytomegalovirus glycoprotein B),Reslizumab (anti-IL-5), Rilotumumab (anti-HGF), Rituximab (MabThera,Rituxanmab, anti-CD20), Robatumumab (anti-IGF-1 receptor), Rontalizumab(anti-IFN-α), Rovelizumab (LeukArrest, anti-CD11, CD18), Ruplizumab(Antova, anti-CD154 (CD40L)), Satumomab (anti-TAG-72), Sevirumab(anti-cytomegalovirus), Sibrotuzumab (anti-FAP), Sifalimumab(anti-IFN-α), Siltuximab (anti-IL-6), Siplizumab (anti-CD2), (Smart)MI95 (anti-CD33), Solanezumab (anti-beta amyloid), Sonepcizumab(anti-sphingosine-1-phosphate), Sontuzumab (anti-episialin), Stamulumab(anti-myostatin), Sulesomab (LeukoScan, (anti-NCA-90 (granulocyteantigen), Tacatuzumab (anti-alpha-fetoprotein), Tadocizumab(anti-integrin α_(IIb)β₃), Talizumab (anti-IgE), Tanezumab (anti-NGF),Taplitumomab (anti-CD19), Tefibazumab (Aurexis, (anti-clumping factorA), Telimomab, Tenatumomab (anti-tenascin C), Teneliximab (anti-CD40),Teplizumab (anti-CD3), TGN1412 (anti-CD28), Ticilimumab (Tremelimumab,(anti-CTLA-4), Tigatuzumab (anti-TRAIL-R₂), TNX-650 (anti-IL-13),Tocilizumab (Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor),Toralizumab (anti-CD154 (CD40L)), Tositumomab (anti-CD20), Trastuzumab(Herceptin, (anti-HER2/neu), Tremelimumab (anti-CTLA-4), Tucotuzumabcelmoleukin (anti-EpCAM), Tuvirumab (anti-hepatitis B virus),Urtoxazumab (anti-Escherichia coli), Ustekinumab (Stelara, anti-IL-12,IL-23), Vapaliximab (anti-AOC3 (VAP-1)), Vedolizumab, (anti-integrinα₄β₇), Veltuzumab (anti-CD20), Vepalimomab (anti-AOC3 (VAP-1),Visilizumab (Nuvion, anti-CD3), Vitaxin (anti-vascular integrin avb3),Volociximab (anti-integrin α₅β₁), Votumumab (HumaSPECT, anti-tumorantigen CTAA16.88), Zalutumumab (HuMax-EGFr, (anti-EGFR), Zanolimumab(HuMax-CD4, anti-CD4), Ziralimumab (anti-CD147 (basigin)), Zolimomab(anti-CD5), Etanercept (Enbrel®), Alefacept (Amevive®), Abatacept(Orencia®), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (Iron RegulatoryProtein 2)], 14G2a (anti-GD2 ganglioside, from Nat. Cancer Inst. formelanoma and solid tumors), J591 (anti-PSMA, Weill Cornell MedicalSchool for prostate cancers), 225.28S [anti-HMW-MAA (High molecularweight-melanoma-associated antigen), Sorin Radiofarmaci S.R.L. (Milan,Italy) for melanoma], COL-1 (anti-CEACAM3, CGM1, from Nat. Cancer Inst.USA for colorectal and gastric cancers), CYT-356 (Oncoltad®, forprostate cancers), HNK20 (OraVax Inc. for respiratory syncytial virus),ImmuRAIT (from Immunomedics for NHL), Lym-1 (anti-HLA-DR10, PeregrinePharm. for Cancers), MAK-195F [anti-TNF (tumor necrosis factor; TNFA,TNF-alpha; TNFSF2), from Abbott/Knoll for Sepsis toxic shock], MEDI-500[T10B9, anti-CD3, TRαβ (T cell receptor alpha/beta), complex, fromMedImmune Inc for Graft-versus-host disease], RING SCAN [anti-TAG 72(tumour associated glycoprotein 72), from Neoprobe Corp. for Breast,Colon and Rectal cancers], Avicidin (anti-EPCAM (epithelial celladhesion molecule), anti-TACSTD1 (Tumor-associated calcium signaltransducer 1), anti-GA733-2 (gastrointestinal tumor-associated protein2), anti-EGP-2 (epithelial glycoprotein 2); anti-KSA; KS1/4 antigen;M4S; tumor antigen 17-1A; CD326, from NeoRx Corp. for Colon, Ovarian,Prostate cancers and NHL]; LymphoCide (Immunomedics, NJ), Smart ID10(Protein Design Labs), Oncolym (Techniclone Inc, CA), Allomune(BioTransplant, CA), anti-VEGF (Genentech, CA); CEAcide (Immunomedics,NJ), IMC-1C11 (ImClone, NJ) and Cetuximab (ImClone, NJ).

Other antibodies as cell binding molecules/ligands include, but are notlimited to, are antibodies against the following antigens:Aminopeptidase N (CD13), Annexin A1, B7-H3 (CD276, various cancers),CA125 (ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6(carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alphafetoprotein (carcinomas), CA242 (colorectal), placental alkalinephosphatase (carcinomas), prostate specific antigen (prostate),prostatic acid phosphatase (prostate), epidermal growth factor(carcinomas), CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma),CD3 epsilon (T cell lymphoma, lung, breast, gastric, ovarian cancers,autoimmune diseases, malignant ascites), CD19 (B cell malignancies),CD20 (non-Hodgkin's lymphoma), CD22 (leukemia, lymphoma, multiplemyeloma, SLE), CD30 (Hodgkin's lymphoma), CD33 (leukemia, autoimmunediseases), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma,leukemia (CLL)), CD51 (Metastatic melanoma, sarcoma), CD52 (leukemia),CD56 (small cell lung cancers, ovarian cancer, Merkel cell carcinoma,and the liquid tumor, multiple myeloma), CD66e (cancers), CD70(metastatic renal cell carcinoma and non-Hodgkin lymphoma), CD74(multiple myeloma), CD80 (lymphoma), CD98 (cancers), mucin (carcinomas),CD221 (solid tumors), CD227 (breast, ovarian cancers), CD262 (NSCLC andother cancers), CD309 (ovarian cancers), CD326 (solid tumors), CEACAM3(colorectal, gastric cancers), CEACAM5 (carcinoembryonic antigen; CEA,CD66e) (breast, colorectal and lung cancers), DLL3 (delta-like-3), DLL4(delta-like-4), EGFR (Epidermal Growth Factor Receptor, variouscancers), CTLA4 (melanoma), CXCR4 (CD184, Heme-oncology, solid tumors),Endoglin (CD105, solid tumors), EPCAM (epithelial cell adhesionmolecule, bladder, head, neck, colon, NHL prostate, and ovariancancers), ERBB2 (Epidermal Growth Factor Receptor 2; lung, breast,prostate cancers), FCGR1 (autoimmune diseases), FOLR (folate receptor,ovarian cancers), GD2 ganglioside (cancers), G-28 (a cell surfaceantigen glyvolipid, melanoma), GD3 idiotype (cancers), Heat shockproteins (cancers), HER1 (lung, stomach cancers), HER2 (breast, lung andovarian cancers), HLA-DR10 (NHL), HLA-DRB (NHL, B cell leukemia), humanchorionic gonadotropin (carcinoma), IGF1R (insulin-like growth factor 1receptor, solid tumors, blood cancers), IL-2 receptor (interleukin 2receptor, T-cell leukemia and lymphomas), IL-6R (interleukin 6 receptor,multiple myeloma, RA, Castleman's disease, IL6 dependent tumors),Integrins (αvβ, α5β1, α6β4,α11β5, αvβ5 for various cancers), MAGE-1(carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE 4(carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma),MS4A1 (membrane-spanning 4-domains subfamily A member 1, Non-Hodgkin's Bcell lymphoma, leukemia), MUC1 or MUC1—KLH (breast, ovarian, cervix,bronchus and gastrointestinal cancer), MUC16 (CA125) (Ovarian cancers),CEA (colorectal), gp100 (melanoma), MART1 (melanoma), MPG (melanoma),MS4A1 (membrane-spanning 4-domains subfamily A, small cell lung cancers,NHL), Nucleolin, Neu oncogene product (carcinomas), P21 (carcinomas),Paratope of anti-(N-glycolylneuraminic acid, Breast, Melanoma cancers),PLAP-like testicular alkaline phosphatase (ovarian, testicular cancers),PSMA (prostate tumors), PSA (prostate), ROBO4, TAG 72 (tumour associatedglycoprotein 72, AML, gastric, colorectal, ovarian cancers), T celltransmembrane protein (cancers), Tie (CD202b), TNFRSF1OB (tumor necrosisfactor receptor superfamily member 1OB, cancers), TNFRSF13B (tumornecrosis factor receptor superfamily member 13B, multiple myeloma, NHL,other cancers, RA and SLE), TPBG (trophoblast glycoprotein, Renal cellcarcinoma), TRAIL-R1 (Tumor necrosis apoprosis Inducing ligand Receptor1,lymphoma, NHL, colorectal, lung cancers), VCAM-1 (CD106, Melanoma),VEGF, VEGF-A, VEGF-2 (CD309) (various cancers). Some other tumorassociated antigens recognized by antibodies have been reviewed (Gerber,et al, mAbs 1:3, 247-53 (2009); Novellino et al, Cancer ImmunolImmunother. 54(3), 187-207 (2005). Franke, et al, Cancer BiotherRadiopharm. 2000, 15, 459-76).

The cell-binding agents, more preferred antibodies, can be any agentsthat are able to against tumor cells, virus infected cells,microorganism infected cells, parasite infected cells, autoimmune cells,activated cells, myeloid cells, activated T-cells, B cells, ormelanocytes. More specifically the cell binding agents can be anyagent/molecule that is able to against any one of the following antigensor receptors: CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e,CD3 g, CD4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD10, CD11a, CD11b,CD11c, CD11d, CD12w, CD14, CD15, CD16, CD16a, CD16b, CDw17, CD18, CD19,CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31,CD32, CD32a, CD32b, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40,CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD46, CD47,CD48, CD49b, CD49c, CD49c, CD49d, CD49f, CD50, CD51, CD52, CD53, CD54,CD55, CD56, CD57, CD58, CD59, CD60, CD60a, CD60b, CD60c, CD61, CD62E,CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66, CD66a, CD66b, CD66c, CD66d,CD66e, CD66f, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75,CD75s, CD76, CD77, CD78, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83,CD84, CD85, CD85a, CD85b, CD85c, CD85d, CD85e, CD85f, CD85 g, CD85 g,CD85i, CD85j, CD85k, CD85m, CD86, CD87, CD88, CD89, CD90, CD91, CD92,CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103,CD104, CD105, CD106, CD107, CD107a, CD107b, CD108, CD109 CD110,CD111,CD112,CD113,CD114,CD115, CD116, CD117,CD118,CD119,CD120 CD120a, CD120b,CD121, CD121a, CD121b, CD122, CD123, CD123a, CD124, CD125, CD126, CD127,CD128, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CD136, CD137,CD138, CD139, CD140, CD140a, CD140b, CD141, CD142, CD143, CD144, CD145,CDw145, CD146, CD147, CD148, CD149, CD150, CD151, CD152, CD153, CD154,CD155, CD156, CD156a, CD156b, CD156c, CD156d, CD157, CD158, CD158a,CD158b1, CD158b2, CD158c, CD158d, CD158e1, CD158e2, CD158f2, CD158 g,CD158h, CD158i, CD158j, CD158k, CD159, CD159a, CD159b, CD159c, CD160,CD161, CD162, CD163, CD164, CD165, CD166, CD167, CD167a, CD167b, CD168,CD169, CD170, CD171, CD172, CD172a, CD172b, CD172g, CD173, CD174, CD175,CD175s, CD176, CD177, CD178, CD179, CD179a, CD179b, CD180, CD181, CD182,CD183, CD184, CD185, CD186, CDw186, CD187, CD188, CD189, CD190, CD191,CD192, CD193, CD194, CD195, CD196, CD197, CD198, CD199, CDw198, CDw199,CD200, CD201, CD202, CD202 (a, b), CD203, CD203c, CD204, CD205, CD206,CD207, CD208, CD209, CD210, CDw210a, CDw210b, CD211, CD212, CD213,CD213a1, CD213a2, CD214, CD215, CD216, CD217, CD218, CD218a, CD218,CD21b9, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228,CD229, CD230, CD231, CD232, CD233, CD234, CD235, CD235a, CD235b, CD236,CD237, CD238, CD239, CD240, CD240ce, CD240d, CD241, CD242, CD243, CD244,CD245, CD246, CD247, CD248, CD249, CD250, CD251, CD252, CD253, CD254,CD255, CD256, CD257, CD258, CD259, CD260, CD261, CD262, CD263, CD264,CD265, CD266, CD267, CD268, CD269, CD270, CD271, CD272, CD273, CD274,CD275, CD276, CD277, CD278, CD279, CD281, CD282, CD283, CD284, CD285,CD286, CD287, CD288, CD289, CD290, CD291, CD292, CD293, CD294, CD295,CD296, CD297, CD298, CD299, CD300, CD300a, CD300b, CD300c, CD301, CD302,CD303, CD304, CD305, CD306, CD307, CD307a, CD307b, CD307c, CD307d,CD307e, CD307f, CD308, CD309, CD310, CD311, CD312, CD313, CD314, CD315,CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD323, CD324, CD325,CD326, CD327, CD328, CD329, CD330, CD331, CD332, CD333, CD334, CD335,CD336, CD337, CD338, CD339, CD340, CD341, CD342, CD343, CD344, CD345,CD346, CD347, CD348, CD349, CD350, CD351, CD352, CD353, CD354, CD355,CD356, CD357, CD358, CD359, CD360, CD361, CD362, CD363, CD364, CD365,CD366, CD367, CD368, CD369, CD370, CD371, CD372, CD373, CD374, CD375,CD376, CD377, CD378, CD379, CD381, CD382, CD383, CD384, CD385, CD386,CD387, CD388, CD389, CRIPTO, CR, CR1, CRGF, CRIPTO, CXCR5, LY64, TDGF1,4-1BB, APO2, ASLG659, BMPR1B, 4-1BB, 5AC, 5T4 (Trophoblast glycoprotein,TPBG, 5T4, Wnt-Activated Inhibitory Factor 1 or WAIF1),Adenocarcinomaantigen, AGS-5, AGS-22M6, Activin receptor-like kinase 1,AFP, AKAP-4, ALK, Alpha intergrin, Alpha v beta6, Amino-peptidase N,Amyloid beta, Androgen receptor, Angiopoietin 2, Angiopoietin 3, AnnexinA1, Anthrax toxin-protective antigen, Anti-transferrin receptor, AOC3(VAP-1), B7-H3, Bacillus anthracisanthrax, BAFF (B-cell activatingfactor), B-lymphoma cell, bcr-abl, Bombesin, BORIS, C5, C242 antigen,CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonicanhydrase 9), CALLA, CanAg, Canis lupus familiaris IL31, Carbonicanhydrase IX, Cardiac myosin, CCL11(C-C motif chemokine 11), CCR4 (C-Cchemokine receptor type 4, CD194), CCR5, CD3E (epsilon), CEA(Carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen),CFD (Factor D), Ch4D5, Cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18),Clumping factor A,CRIPTO, FCSF1R (Colony stimulating factor 1 receptor,CD115), CSF2 (colony stimulating factor 2, Granulocyte-macrophagecolony-stimulating factor (GM-CSF)), CTLA4 (cytotoxic T-lymphocyteassociated protein 4), CTAA16.88 tumor antigen, CXCR4 (CD184),C-X-Cchemokine receptor type 4, cyclic ADP ribose hydrolase, Cyclin B1,CYP1B1, Cytomegalovirus, Cytomegalovirus glycoprotein B, Dabigatran,DLL3 (delta-like-ligand 3), DLL4 (delta-like-ligand 4), DPP4(Dipeptidyl-peptidase 4), DR5 (Death receptor 5), E. coli shigatoxintype-1, E. coli shiga toxintype-2, ED-B, EGFL7 (EGF-likedomain-containing protein 7), EGFR, EGFRII, EGFRvIII, Endoglin (CD105),Endothelin B receptor, Endotoxin, EpCAM (epithelial cell adhesionmolecule), EphA2, Episialin, ERBB2 (Epidermal Growth Factor Receptor 2),ERBB3, ERG (TMPRSS2 ETS fusion gene), Escherichia coli,ETV6-AML, FAP(Fibroblast activation protein alpha), FCGR1, alpha-Fetoprotein, FibrinII, beta chain, Fibronectin extra domain-B, FOLR (folate receptor),Folate receptor alpha, Folate hydrolase, Fos-related antigen 1, Fprotein of respiratory syncytial virus, Frizzled receptor, FucosylGM1,GD2 ganglioside, G-28 (a cell surface antigen glyvolipid), GD3idiotype, GloboH, Glypican 3, N-glycolylneuraminic acid, GM3, GMCSFreceptor α-chain, Growth differentiation factor 8, GP100, GPNMB(Transmembrane glycoprotein NMB), GUCY2C (Guanylate cyclase 2C, guanylylcyclase C(GC-C), intestinal Guanylate cyclase, Guanylate cyclase-Creceptor, Heat-stable enterotoxin receptor (hSTAR)), Heat shockproteins, Hemagglutinin, Hepatitis B surface antigen, Hepatitis B virus,HER1 (human epidermal growth factor receptor 1), HER2, HER2/neu, HER3(ERBB-3), IgG4, HGF/SF (Hepatocyte growth factor/scatter factor), HHGFR,HIV-1, Histone complex, HLA-DR (human leukocyte antigen), HLA-DR10,HLA-DRB, HMWMAA, Human chorionic gonadotropin, HNGF, Human scatterfactor receptor kinase, HPV E6/E7, Hsp90, hTERT, ICAM-1 (IntercellularAdhesion Molecule 1), Idiotype, IGF1R (IGF-1, insulin-like growth factor1 receptor), IGHE, IFN-γ, Influeza hemag-glutinin, IgE, Fc region, IGHE,IL-1, IL-2 receptor (interleukin 2 receptor), IL-4, IL-5, IL-6, IL-6R(interleukin 6 receptor), IL-9, IL-10, IL-12, IL-13, IL-17, IL-17A,IL-20, IL-22, IL-23, IL31RA, ILGF2 (Insulin-like growth factor 2),Integrins (α4, α_(IIb)β3, αvβ, α₄β₇, α5β1, α6β4, α7β7, α11β3, αvβ5),Interferon gamma-induced protein, ITGA2, ITGB2, KIR2D, LCK, Le,Legumain, Lewis-Y antigen, LFA-1(Lymphocyte function-associated antigen1, CDI1a), LHRH, LINGO-1, Lipoteichoic acid, LIVIA, LMP2, LTA, MAD-CT-1,MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE A1, MAGE A3, MAGE 4, MART1,MCP-1, MIF (Macrophage migration inhibitory factor, orglycosylation-inhibiting factor (GIF)), MS4A1 (membrane-spanning4-domains subfamily A member 1), MSLN (meso-thelin), MUC1(Mucin 1, cellsurface associated (MUC1) or polymorphic epithelial mucin (PEM)),MUC1—KLH, MUC16 (CAI25), MCP1(monocyte chemotactic protein 1),MelanA/MART1, ML-IAP, MPG, MS4A1 (membrane-spanning 4-domains subfamilyA), MYCN, Myelin-associated glycoprotein, Myostatin, NA17, NARP-1,NCA-90 (granulocyte antigen), Nectin-4 (ASG-22ME), NGF, Neuralapoptosis-regulated proteinase 1, NOGO-A, Notch receptor, Nucleolin, Neuoncogene product, NY-BR-1, NY-ESO—1, OX-40, OxLDL (Oxidized low-densitylipoprotein), OY-TES1,P21, p53 nonmutant, P97, Page4, PAP, Paratope ofanti-(N-glycolylneuraminic acid), PAX3, PAX5, PCSK9, PDCD1 (PD-1,Programmed cell death protein 1,CD279), PDGF-Rα (Alpha-typeplatelet-derived growth factor receptor), PDGFR-β, PDL-1, PLAC1,PLAP-like testicular alkaline phosphatase, Platelet-derived growthfactor receptor beta, Phosphate-sodium co-transporter, PMEL 17,Polysialic acid, Proteinase3 (PRI), Prostatic carcinoma, PS(Phosphatidylserine), Prostatic carcinoma cells, Pseudomonas aeruginosa,PSMA, PSA, PSCA, Rabies virus glycoprotein, RHD (Rh polypeptide 1(RhPI), CD240), Rhesus factor, RANKL, RhoC, Ras mutant, RGS5, ROBO4,Respiratory syncytial virus, RON, Sarcoma translocationbreakpoints,SART3, Sclerostin, SLAMF7 (SLAM family member 7), SelectinP, SDC1 (Syndecan 1), sLe(a), Somatomedin C, SIP(Sphingosine-1-phosphate), Somatostatin, Sperm protein 17, SSX2, STEAPI(six-transmembrane epithelial antigen of the prostate 1), STEAP2, STn,TAG-72 (tumor associated glycoprotein 72), Survivin, T-cell receptor, Tcell transmembrane protein, TEM1 (Tumor endothelial marker 1), TENB2,Tenascin C (TN-C), TGF-α, TGF-β (Transforming growth factor beta),TGF-β1, TGF-β2 (Transforming growth factor-beta 2), Tie (CD202b), Tie2,TIM-1 (CDX-014), Tn, TNF, TNF-α, TNFRSF8, TNFRSF1OB (tumor necrosisfactor receptor superfamily member 10B), TNFRSF13B (tumor necrosisfactor receptor superfamily member 13B), TPBG (trophoblastglycoprotein), TRAIL-R1 (Tumor necrosis apoprosis Inducing ligandReceptor 1), TRAILR2 (Death receptor 5 (DR5)), tumor-associated calciumsignal transducer 2, tumor specific glycosylation ofMUCI, TWEAKreceptor, TYRP1 (glycoprotein 75), TROP-2, TRP-2, Tyrosinase, VCAM-1(CD106), VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2, or vimentin,WTi, XAGE 1, or cells expressing any insulin growth factor receptors, orany epidermal growth factor receptors.

In another specific embodiment, the cell-binding molecule can be aligand or a receptor agonist selected from: folate derivatives (bindingto the folate receptor, a protein over-expressed in ovarian cancer andin other malignancies) (Low, P. S. et al 2008, Acc. Chem. Res. 41,120-9); glutamic acid urea derivatives (binding to the prostate specificmembrane antigen, a surface marker of prostate cancer cells) (Hillier,S. M.et al, 2009, Cancer Res. 69, 6932-40); Somatostatin (also known asgrowth hormone-inhibiting hormone (GHIH) or sornatotropinrelease-inhibiting factor (SRIF)) or somatotropin release-inhibitinghormone) and its analogues such as octreotide (Sandostatin) andlanreotide (Somatuline) (particularly for neuroendocrine tumors,GH-producing pituitary adenoma, paraganglioma, nonfunctioning pituitaryadenoma, pheochromocytomas) (Ginj, M., et al, 2006, Proc. Natl. Acad.Sci. U.S.A. 103, 16436-41); Somatostatin receptor subtypes (sstl, sst2,sst3, sst4, and sst5) in GH-secreting pituitaryadenomas (Reubi J. C.,Landolt, A. M. 1984 J. Clin. Endocrinol Metab 59: 1148-51; Reubi J. C.,Landolt A. M. 1987 J Clin Endocrinol Metab 65: 65-73; Moyse E, et al, JClin Endocrinol Metab 61: 98-103), gastroenteropancreatic tumors (ReubiJ. C., et al, 1987 J Clin Endocrinol Metab 65: 1127-34; Reubi, J. C, etal, 1990 Cancer Res 50: 5969-77), pheochromocytomas (Epel-baum J, et al1995 J Clin Endocrinol Metab 80:1837-44; Reubi J. C., et al, 1992 J ClinEndocrinol Metab 74: 1082-9), neuroblastomas (Prevost G, 1996Neuroendocrinology 63:188-197; Moertel, C. L, et al 1994 Am J Clin Path102:752-756), medullary thyroid cancers (Reubi, J. C, et al 1991 LabInvest 64:567-573) small cell lung cancers (Sagman U, et al, 1990 Cancer66:2129-2133), meningiomas, medulloblastomas, or gliomas (Reubi J. C.,et al 1986 J Clin Endocrinol Metab 63: 433-8; Reubi J. C., et al 1987Cancer Res 47: 5758-64; Fruhwald, M. C, et al 1999 Pediatr Res 45:697-708), breast carcinomas (Reubi J. C., et al 1990 Int J Cancer 46:416-20; Srkalovic G, et al 1990 J Clin Endocrinol Metab 70: 661-669),lymphomas (Reubi J. C., et al 1992, Int J Cancer50: 895-900), renal cellcancers (Reubi J. C., et al 1992, Cancer Res 52: 6074-6078), mesenchymaltumors (Reubi J. C., et al 1996 Cancer Res 56: 1922-31), prostatic(Reubi J. C., et al 1995, J. Clin.

Endocrinol Metab 80: 2806-14; et al 1989, Prostate 14:191-208; Halmos G,et al J. Clin. Endo-crinol Metab 85: 2564-71), ovarian (Halmos, G, etal, 2000 J Clin Endocrinol Metab 85: 3509-12; Reubi J. C., et al 1991 AmJ Pathol 138:1267-72), gastric (Reubi J. C., et al 1999, Int J Cancer81: 376-86; Miller, G. V, 1992 Br J Cancer 66: 391-95), hepatocellular(Kouroumalis E, et al 1998 Gut 42: 442-7; Reubi J. C., et al 1999 Gut45: 66-774) and nasopharyngeal carcinomas (Loh K. S, et al, 2002Virchows Arch 441: 444-8); Aromatic sulfonamides (specific to carbonicanhydrase IX) (a marker of hypoxia and of renal cell carcinoma) (Neri,D., et al, Nat. Rev. Drug Discov. 2011, 10, 767-7); Pituitary adenylatecyclase activating peptides (PACAP) (PAC1) for pheochromocytomas andparagangliomas; Vasoactive intestinal peptides (VIP)and their receptorsubtypes (VPAC1, VPAC2); α-Melanocyte-stimulating hormone (α-MSH)receptors; Cholecystokinin (CCK)/gastrin receptors and their receptorsubtypes (CCK1 (formerly CCK-A) and CCK2;Bombesin(Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂)/gastrin-releasingpeptide (GRP) and their receptor subtypes (BB1, GRP receptor subtype(BB2), the BB3 and BB4) (Ohlsson, B., et al, 1999, Scand. J.Gastroenterology 34(12): 1224-9; Weber, H. C., 2009, Cur. Opin. Endocri.Diab. Obesity 16(1): 66-71, Gonzalez N. et al. 2008, Cur. Opin. Endocri.Diab. Obesity 15(1), 58-64); Neurotensin receptors and its receptorsubtypes(NTR1, NTR2, NTR3); Substance P receptors and their receptorsubtypes(such as NK1 receptor for Glial tumors, Hennig I. M., et al 1995Int. J. Cancer 61, 786-792); Neuropeptide Y (NPY) receptors and itsreceptor subtypes (Y1-Y6); Homing Peptides include RGD (Arg-Gly-Asp),NGR (Asn-Gly-Arg), the dimeric and multimeric cyclic RGD peptides (e.g.cRGDfV) (Laakkonen P, Vuorinen K. 2010, Integr Biol (Camb). 2(7-8):326-337; Chen K, Chen X. 2011, Theranostics. 1:189-200; Garanger E, etal, Anti-Cancer Agents Med Chem. 7 (5): 552-558; Kerr, J. S. et al,Anticancer Research, 19(2A), 959-968; Thumshim, G, et al, 2003 Chem.Eur. J. 9, 2717-2725), and TAASGVRSMH or LTLRWVGLMS (chondroitin sulfateproteoglycan NG2 receptor) and F3 peptides (31 amino acid peptide thatbinds to cell surface-expressed nucleolin receptor) (Zitzmann, S., 2002Cancer Res., 62, 18, pp. 5139-5143, Temminga, K., 2005, Drug ResistanceUpdates, 8, 381-402; P. Laakkonen and K. Vuorinen, 2010 IntegrativeBiol, 2(7-8), 326-337; M. A. Burg, 1999 Cancer Res., 59(12), 2869-2874;K. Porkka, et al 2002, Proc. Nat.

Acad. Sci. USA 99(11), 7444-9); Cell Penetrating Peptides (CPPs) (NakaseI, et al, 2012, J. Control Release. 159(2),181-188); Peptide Hormones,such as luteinizing hormone-releasing hormone (LHRH) agonists andantagonists, and gonadotropin-releasing hormone (GnRH) agonist, acts bytargeting follicle stimulating hormone (FSH) and luteinising hormone(LH), as well as testosterone production, e.g. buserelin(Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-NHEt), Gonadorelin(Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂), Goserelin(Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-AzGly-NH₂), Histrelin(Pyr-His-Trp-Ser-Tyr-D-His(N-benzyl)-Leu-Arg-Pro-NHEt), leuprolide(Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt), Nafarelin(Pyr-His-Trp-Ser-Tyr-2Nal-Leu-Arg-Pro-Gly-NH₂), Triptorelin(Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH₂), Nafarelin, Deslorelin,Abarelix(Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu-isopropylLys-Pro-DAla-NH₂),Cetrorelix(Ac-D-2Nal-D-4-chloro-Phe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH₂),Degarelix(Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-4-aminoPhe(L-hydroorotyl)-D-4-aminoPhe(carba-moyl)-Leu-isopropylLys-Pro-D-Ala-NH₂),and Ganirelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-(N9,N10-diethyl)-homoArg-Leu-(N9, N10-diethyl)-homoArg-Pro-D-Ala-NH₂)(Thundimadathil, J., J. Amino Acids, 2012, 967347,doi:10.1155/2012/967347; Boccon-Gibod, L_; et al, 2011, TherapeuticAdvances in Urology 3(3): 127-140; Debruyne, F., 2006, Future Oncology,2(6), 677-696; Schally A. V; Nagy, A. 1999 Eur J Endocrinol 141:1-14;Koppan M, et al 1999 Prostate 38:151-158); and Pattern RecognitionReceptors (PRRs), such as Toll-like receptors (TLRs), C-type lectins andNodlike Receptors (NLRs) (Fukata, M., et al, 2009, Semin.

Immunol. 21, 242-253; Maisonneuve, C., et al, 2014, Proc. Natl. Acad.Sci. U.S.A 111, 1-6; Botos, I., et al, 2011, Structure 19, 447-459;Means, T. K., et al, 2000, Life Sci. 68, 241-258) that range in sizefrom small molecules (imiquimod, guanisine and adenosine analogs)tolarge and complex biomacromolecules such as lipopolysaccharide (LPS),nucleic acids (CpG DNA, polyL:C) and lipopeptides (Pam3CSK4) (Kasturi,S. P., et al, 2011, Nature 470, 543-547; Lane, T., 2001, J. R. Soc. Med.94, 316; Hotz, C., and Bourquin, C., 2012, Oncoimmunology 1, 227-228;Dudek, A. Z., et al, 2007, Clin. Cancer Res. 13, 7119-25); Calcitoninreceptors which is a 32-amino-acid neuropeptide involved in theregulation of calcium levels largely through its effects on osteoclastsand on the kidney (Zaidi M, et al, 1990 Crit Rev Clin Lab Sci 28,109-174; Gorn, A. H., et al 1995 J Clin Invest 95:2680-91); And integrinreceptors and their receptor subtypes (such as uvDi, αvβ, αvβ₅, αvρ₆,α₆β₄, α₇β₁, α_(IIb)β₃, etc.) which generally play important roles inangiogenesis are expressed on the surfaces of a variety of cells, inparticular, of osteoclasts, endothelial cells and tumor cells(Ruoslahti, E. et al, 1994 Cell 77, 477-8; Albelda, S. M. et al, 1990Cancer Res., 50, 6757-64). Short peptides, GRGDSPK and Cyclic RGDpentapeptides, such as cyclo(RGDfV) (L1) and its derives[cyclo(-N(Me)R-GDfV), cyclo(R-Sar-DfV), cyclo-(RG-N(Me)D-fV),cyclo(RGD-N(Me)f-V), cyclo(RGDf-N(Me)V-)(Cilengitide)]have shown highbinding affinities of the intergrin receptors (Dechantsreiter, M. A. etal, 1999 J.

Med. Chem. 42, 3033-40, Goodman, S. L., et al, 2002 J. Med. Chem. 45,1045-51).

The cell-binding molecule/ligands or cell receptor agonists can beIg-based and non-Ig-based protein scaffold molecules. The Ig-Basedscaffolds can be selected, but not limited, from Nanobody (a derivativeof VHH (camelid Ig)) (Muyldermans S., 2013 Annu Rev Biochem. 82,775-97); Domain antibodies (dAb, a derivative of VH or VL domain) (Holt,L. J, et al, 2003, Trends Biotechnol. 21, 484-90); Bispecific T cellEngager (BiTE, a bispecific diabody) (Baeuerle, P. A, et al, 2009, Curr.Opin. Mol. Ther. 11, 22-30); Dual Affinity ReTargeting (DART, abispecific diabody) (Moore P. A. P, et al. 2011, Blood 117(17),4542-51); Tetravalent tandem antibodies (TandAb, a dimerized bispecificdiabody) (Cochlovius, B, et al. 2000, Cancer Res. 60(16):4336-4341). TheNon-Ig scaffolds can be selected, but not limited, from Anticalin (aderivative of Lipocalins) (Skerra A. 2008, FEBS J., 275(11): 2677-83;Beste G, et al, 1999 Proc. Nat. Acad. USA. 96(5):1898-903; Skerra, A.2000 Biochim Biophys Acta, 1482(1-2): 337-50; Skerra, A. 2007, Curr OpinBiotechnol. 18(4): 295-304; Skerra, A. 2008, FEBS J. 275(11):2677-83);Adnectins (10th FN3 (Fibronectin)) (Koide, A, et al, 1998 J. Mol. Biol.,284(4):1141-51; Baton V, 2002, Protein Eng. 15(12): 1015-20; Tolcher, A.W, 2011, Clin. Cancer Res. 17(2): 363-71; Hackel, B. J, 2010, ProteinEng. Des. Sel. 23(4): 211-19); Designed Ankyrin Repeat Proteins(DARPins) (a derivative of ankrin repeat (AR) proteins) (Boersma, Y. L,et al, 2011 Curr Opin Biotechnol. 22(6): 849-57), e.g. DARPin C9, DARPinEc4 and DARPin E69_LZ3_E01 (Winkler J, et al, 2009 Mol Cancer Ther.8(9), 2674-83; Patricia M-K. M., et al, Clin Cancer Res. 2011;17(1):100-10; Boersma Y. L, et al, 2011 J. Biol. Chem. 286(48),41273-85); Avimers (a domain A/low-density lipoprotein (LDL) receptor)(Boersma Y. L, 2011 J. Biol. Chem. 286(48): 41273-41285; Silverman J, etal, 2005 Nat. Biotechnol., 23(12):1556-61).

Examples of the small molecule structures of the cell-bindingmolecules/ligands or cell receptor agonists of the patent applicationare the following: LBO1 (Folate), LB02 (PMSA ligand), LB03 (PMSAligand), LB04 (PMSA ligand), LB05 (Somatostatin), LB06 (Somatostatin),LB07 (Octreotide, a Somatostatin analog), LB08 (Lanreotide, aSomatostatin analog), LB09 (Vapreotide (Sanvar), a Somatostatin analog),LB10 (CAIX ligand), LB11 (CAIX ligand), LB12 (Gastrin releasing peptidereceptor (GRPr), MBA), LB13 (luteinizing hormone-releasing hormone(LH-RH) ligand and GnRH), LB14 (luteinizing hormone-releasing hormone(LH-RH) and GnRH ligand), LB15 (GnRH antagonist, Abarelix), LB16(cobalamin, vitamin B12 analog), LB17 (cobalamin, vitamin B12 analog),LB18 (for α_(v)β₃ integrin receptor, cyclic RGD pentapeptide), LB19(hetero-bivalent peptide ligand for VEGF receptor), LB20 (Neuromedin B),LB21 (bombesin for a G-protein coupled receptor), LB22 (TLR₂ for aToll-like receptor,), LB23 (for an androgen receptor), LB24(Cilengitide/cyclo(-RGDfV-) for an α_(v) intergrin receptor, LB23(Fludrocortisone), LB25 (Rifabutin analog), LB26 (Rifabutin analog),LB27 (Rifabutin analog), LB28 (Fludrocortisone), LB29 (Dexamethasone),LB30 (fluticasone propionate), LB31 (Beclometasone dipropionate), LB32(Triamcinolone acetonide), LB33 (Prednisone), LB34 (Prednisolone), LB35(Methylprednisolone), LB36 (Betamethasone), LB37 (Irinotecan analog),LB38 (Crizotinib analog), LB39 (Bortezomib analog), LB40 (Carfilzomibanalog), LB41 (Carfilzomib analog), LB42 (Leuprolide analog), LB43(Triptorelin analog), LB44 (Clindamycin), LB45 (Liraglutide analog),LB46 (Semaglutide analog), LB47 (Retapamulin analog), LB48 (Indibulinanalog), LB49 (Vinblastine analog), LB50 (Lixisenatide analog), LB51(Osimertinib analog), LB52 (a neucleoside analog), LB53 (Erlotinibanalog) and LB54 (Lapatinib analog) which are shown in the followingstructures:

wherein

is the site to link the side chain linker of the present patent; X₄,andY₁ are independently O, NH, NHNH, NR₁, S, C(O)O, C(O)NH, OC(O)NH,OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N(R₁), N(R₁)C(O)N(R₁), CH₂, C(O)NHNHC(O)and C(O)NR₁; X₁ is H, CH₂, OH, O, C(O), C(O)NH, C(O)N(R₁), R₁, NHR₁,NR₁, C(O)R₁ or C(O)O; X₅ is H, CH₃, F, or C₁; M₁ and M₂ areindependently H, Na, K, Ca, Mg, NH₄, N(R₁R₂R₃R⁴); R₁, R₂, R₃ and R⁴ aredefined in Formula (I);

Application of the Conjugate

In a specific embodiment, the cell-binding ligand-drug conjugates viathe side chain linkers of this invention are used for the targetedtreatment of cancers. The targeted cancers include, but are not limited,Adrenocortical Carcinoma, Anal Cancer, Bladder Cancer, Brain Tumor(Adult, Brain Stem Glioma, Childhood, Cerebellar Astrocytoma, CerebralAstrocytoma, Ependymoma, Medulloblastoma, Supratentorial PrimitiveNeuroectodermal and Pineal Tumors, Visual Pathway and HypothalamicGlioma), Breast Cancer, Carcinoid Tumor, Gastrointestinal, Carcinoma ofUnknown Primary, Cervical Cancer, Colon Cancer, Endometrial Cancer,Esophageal Cancer, Extrahepatic Bile Duct Cancer, Ewings Family ofTumors (PNET), Extracranial Germ Cell Tumor, Eye Cancer, IntraocularMelanoma, Gallbladder Cancer, Gastric Cancer (Stomach), Germ Cell Tumor,Extragonadal, Gestational Trophoblastic Tumor, Head and Neck Cancer,Hypopharyngeal Cancer, Islet Cell Carcinoma, Kidney Cancer (renal cellcancer), Laryngeal Cancer, Leukemia (Acute Lymphoblastic, Acute Myeloid,Chronic Lymphocytic, Chronic Myelogenous, Hairy Cell), Lip and OralCavity Cancer, Liver Cancer, Lung Cancer (Non-Small Cell, Small Cell,Lymphoma (AIDS-Related, Central Nervous System, Cutaneous T-Cell,Hodgkin's Disease, Non-Hodgkin's Disease, Malignant Mesothelioma,Melanoma, Merkel Cell Carcinoma, Metasatic Squamous Neck Cancer withOccult Primary, Multiple Myeloma, and Other Plasma Cell Neoplasms,Mycosis Fungoides, Myelodysplastic Syndrome, MyeloproliferativeDisorders, Nasopharyngeal Cancer, Neuroblastoma, Oral Cancer,Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer (Epithelial, GermCell Tumor, Low Malignant Potential Tumor), Pancreatic Cancer (Exocrine,Islet Cell Carcinoma), Paranasal Sinus and Nasal Cavity Cancer,Parathyroid Cancer, Penile Cancer, Pheochromocytoma Cancer, PituitaryCancer, Plasma Cell Neoplasm, Prostate Cancer Rhabdomyosarcoma, RectalCancer, Renal Cell Cancer (kidney cancer), Renal Pelvis and Ureter(Transitional Cell), Salivary Gland Cancer, Sezary Syndrome, SkinCancer, Skin Cancer (Cutaneous T-Cell Lymphoma, Kaposi's Sarcoma,Melanoma), Small Intestine Cancer, Soft Tissue Sarcoma, Stomach Cancer,Testicular Cancer, Thymoma (Malignant), Thyroid Cancer, Urethral Cancer,Uterine Cancer (Sarcoma), Unusual Cancer of Childhood, Vaginal Cancer,Vulvar Cancer, Wilms' Tumor.

In another specific embodiment, the cell-binding-drug conjugates of thisinvention are used in accordance with the compositions and methods forthe treatment or prevention of an autoimmune disease. The autoimmunediseases include, but are not limited, Achlorhydra Autoimmune ActiveChronic Hepatitis, Acute Disseminated Encephalomyelitis, Acutehemorrhagic leukoencephalitis, Addison's Disease, Agammaglobulinemia,Alopecia areata, Amyotrophic Lateral Sclerosis, Ankylosing Spondylitis,Anti-GBM/TBM Nephritis, Antiphospholipid syndrome, Antisynthetasesyndrome, Arthritis, Atopic allergy, Atopic Dermatitis, AutoimmuneAplastic Anemia, Autoimmune cardiomyopathy, Autoimmune hemolytic anemia,Autoimmune hepatitis, Autoimmune inner ear disease, Autoimmunelymphoproliferative syndrome, Autoimmune peripheral neuropathy,Autoimmune pancreatitis, Autoimmune polyendocrine syndrome Types I, II,& III, Autoimmune progesterone dermatitis, Autoimmune thrombocytopenicpurpura, Autoimmune uveitis, Balo disease/Balo concentric sclerosis,Bechets Syndrome, Berger's disease, Bickerstaff s encephalitis, Blausyndrome, Bullous Pemphigoid, Castleman's disease, Chagas disease,Chronic Fatigue Immune Dysfunction Syndrome, Chronic inflammatorydemyelinating polyneuropathy, Chronic recurrent multifocal ostomyelitis,Chronic lyme disease, Chronic obstructive pulmonary disease,Churg-Strauss syndrome, Cicatricial Pemphigoid, Coeliac Disease, Cogansyndrome, Cold agglutinin disease, Complement component 2 deficiency,Cranial arteritis, CREST syndrome, Crohns Disease (a type of idiopathicinflammatory bowel diseases), Cushing's Syndrome, Cutaneousleukocytoclastic angiitis, Dego's disease, Dercum's disease, Dermatitisherpetiformis, Dermatomyositis, Diabetes mellitus type 1, Diffusecutaneous systemic sclerosis, Dressler's syndrome, Discoid lupuserythematosus, Eczema, Endometriosis, Enthesitis-related arthritis,Eosinophilic fasciitis, Epidermolysis bullosa acquisita, Erythemanodosum, Essential mixed cryoglobulinemia, Evan's syndrome,Fibrodysplasia ossificans progressiva, Fibromyalgia, Fibromyositis,Fibrosing aveolitis, Gastritis, Gastrointestinal pemphigoid, Giant cellarteritis, Glomerulonephritis, Goodpasture's syndrome, Graves' disease,Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto'sthyroiditis, Haemolytic anaemia, Henoch-Schonlein purpura, Herpesgestationis, Hidradenitis suppurativa, Hughes syndrome (SeeAntiphospholipid syndrome), Hypogamma-globulinemia, IdiopathicInflammatory Demyelinating Diseases, Idiopathic pulmonary fibrosis,Idiopathic thrombocytopenic purpura (See Autoimmune thrombocytopenicpurpura), IgA nephropathy (Also Berger's disease), Inclusion bodymyositis, Inflammatory demyelinating polyneuopathy, Interstitialcystitis, Irritable Bowel Syndrome, Juvenile idiopathic arthritis,Juvenile rheumatoid arthritis, Kawasaki's Disease, Lambert-Eatonmyasthenic syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichensclerosus, Linear IgA disease (LAD), Lou Gehrig's Disease (AlsoAmyotrophic lateral sclerosis), Lupoid hepatitis, Lupus erythematosus,Majeed syndrome, Meniere's disease, Microscopic polyangiitis,Miller-Fisher syndrome, Mixed Connective Tissue Disease, Morphea,Mucha-Habermann disease, Muckle-Wells syndrome, Multiple Myeloma,Multiple Sclerosis, Myasthenia gravis, Myositis, Narcolepsy,Neuromyelitis optica (Devic's Disease), Neuromyotonia, Occularcicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord thyroiditis,Palindromic rheumatism, PANDAS (Pediatric Autoimmune NeuropsychiatricDisorders Associated with Streptococcus), Paraneoplastic cerebellardegeneration, Paroxysmal nocturnal hemoglobinuria, Parry Rombergsyndrome, Parsonnage-Turner syndrome, Pars planitis, Pemphigus,Pemphigus vulgaris, Pernicious anaemia, Perivenous encephalomyelitis,POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica,Polymyositis, Primary biliary cirrhosis, Primary sclerosing cholangitis,Progressive inflammatory neuropathy, Psoriasis, Psoriatic Arthritis,Pyoderma gangrenosum, Pure red cell aplasia, Rasmussen's encephalitis,Raynaud phenomenon, Relapsing polychondritis, Reiter's syndrome,Restless leg syndrome, Retroperitoneal fibrosis, Rheumatoid arthritis,Rheumatoid fever, Sarcoidosis, Schizophrenia, Schmidt syndrome,Schnitzler syndrome, Scleritis, Scleroderma, Sjögren's syndrome,Spondyloarthropathy, Sticky blood syndrome, Still's Disease, Stiffperson syndrome, Subacute bacterial endocarditis, Susac's syndrome,Sweet syndrome, Sydenham Chorea, Sympathetic ophthalmia, Takayasu'sarteritis, Temporal arteritis (giant cell arteritis), Tolosa-Huntsyndrome, Transverse Myelitis, Ulcerative Colitis (a type of idiopathicinflammatory bowel diseases), Undifferentiated connective tissuedisease, Undifferentiated spondyloarthropathy, Vasculitis, Vitiligo,Wegener's granulomatosis, Wilson's syndrome, Wiskott-Aldrich syndrome

In another specific embodiment, a binding molecule used for theconjugate via the side chain-linkers of this invention for the treatmentor prevention of an autoimmune disease can be, but are not limited to,anti-elastin antibody; Abys against epithelial cells antibody;Anti-Basement Membrane Collagen Type IV Protein antibody; Anti-NuclearAntibody; Anti ds DNA; Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG;anti-celiac antibody; Anti Phospholipid Antibody IgK, IgG; Anti SMAntibody; Anti Mitochondrial Antibody; Thyroid Antibody; MicrosomalAntibody, T-cells antibody; Thyroglobulin Antibody, Anti SCL-70;Anti-Jo; Anti-U.sub.1RNP; Anti-La/SSB; Anti SSA; Anti SSB; Anti PeritalCells Antibody; Anti Histones; Anti RNP; C-ANCA; P-ANCA; Anticentromere; Anti-Fibrillarin, and Anti GBM Antibody, Anti-gangliosideantibody; Anti-Desmogein 3 antibody; Anti-p62 antibody; Anti-sp100antibody; Anti-Mitochondrial(M2) antibody; Rheumatoid factor antibody;Anti-MCV antibody; Anti-topoisomerase antibody; Anti-neutrophilcytoplasmic(cANCA) antibody.

In certain preferred embodiments, the binding molecule for the conjugatein the present invention, can bind to both a receptor and a receptorcomplex expressed on an activated lymphocyte which is associated with anautoimmune disease. The receptor or receptor complex can comprise animmunoglobulin gene superfamily member (e.g. CD2, CD3, CD4, CD8, CD19,CD20, CD22, CD28, CD30, CD33, CD37, CD38, CD56, CD70, CD79, CD79b, CD90,CD125, CD137, CD138, CD147, CD152/CTLA-4, PD-1, or ICOS), a TNF receptorsuperfamily member (e.g. CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB,INF-R₁, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R₁,TRAIL-R₂, TRAIL-R₃, TRAIL-R⁴, and APO—3), an integrin, a cytokinereceptor, a chemokine receptor, a major histocompatibility protein, alectin (C-type, S-type, or I-type), or a complement control protein.

In another specific embodiment, useful cell binding ligands that areimmunospecific for a viral or a microbial antigen are humanized or humanmonoclonal antibodies. As used herein, the term “viral antigen”includes, but is not limited to, any viral peptide, polypeptide protein(e.g. HIV gp120, HIV nef, RSV F glycoprotein, influenza virusneuraminidase, influenza virus hemagglutinin, HTLV tax, herpes simplexvirus glycoprotein (e.g. gB, gC, gD, and gE) and hepatitis B surfaceantigen) that is capable of eliciting an immune response. As usedherein, the term “microbial antigen” includes, but is not limited to,any microbial peptide, polypeptide, protein, saccharide, polysaccharide,or lipid molecule (e.g., a bacteria, fungi, pathogenic protozoa, oryeast polypeptides including, e.g., LPS and capsular polysaccharide 5/8)that is capable of eliciting an immune response. Examples of antibodiesavailable 1 for the viral or microbial infection include, but are notlimited to, Palivizumab which is a humanized anti-respiratory syncytialvirus monoclonal antibody for the treatment of RSV infection; PR0542which is a CD4 fusion antibody for the treatment of HIV infection;Ostavir which is a human antibody for the treatment of hepatitis Bvirus; PROTVIR which is a humanized IgG.sub.1 antibody for the treatmentof cytomegalovirus; and anti-LPS antibodies.

The cell binding molecules-drug conjugates via the side chain-linkers ofthis invention can be used in the treatment of infectious diseases.These infectious diseases include, but are not limited to, Acinetobacterinfections, Actinomycosis, African sleeping sickness (Africantrypanosomiasis), AIDS (Acquired immune deficiency syndrome), Amebiasis,Anaplasmosis, Anthrax, Arcano-bacterium haemolyticum infection,Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovirusinfection, Babesiosis, Bacillus cereus infection, Bacterial pneumonia,Bacterial vaginosis, Bacteroides infection, Balantidiasis, Baylisascarisinfection, BK virus infection, Black piedra, Blastocystis hominisinfection, Blastomycosis, Bolivian hemorrhagic fever, Borreliainfection, Botulism (and Infant botulism), Brazilian hemorrhagic fever,Brucellosis, Burkholderia infection, Buruli ulcer, Calicivirus infection(Norovirus and Sapovirus), Campylobacteriosis, Candidiasis (Moniliasis;Thrush), Cat-scratch disease, Cellulitis, Chagas Disease (Americantrypanosomiasis), Chancroid, Chickenpox, Chlamydia, Chlamydophilapneumoniae infection, Cholera, Chromoblastomycosis, Clonorchiasis,Clostridium difficile infection, Coccidioido-mycosis, Colorado tickfever, Common cold (Acute viral rhinopharyngitis; Acute coryza),Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever,Cryptococcosis, Cryptosporidiosis, Cutaneous larva migrans,Cyclosporiasis, Cysticercosis, Cytomegalovirus infection, Dengue fever,Dientamoebiasis, Diphtheria, Diphyllobothriasis, Dracunculiasis, Ebolahemorrhagic fever, Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworminfection), Enterococcus infection, Enterovirus infection, Epidemictyphus, Erythema infectiosum (Fifth disease), Exanthem subitum,Fasciolopsiasis, Fasciolosis, Fatal familial insomnia, Filariasis, Foodpoisoning by Clostridium perfringens, Free-living amebic infection,Fusobacterium infection, Gas gangrene (Clostridial myonecrosis),Geotrichosis, Gerstmann-Straussler-Scheinker syndrome, Giardiasis,Glanders, Gnathosto-miasis, Gonorrhea, Granuloma inguinale(Donovanosis), Group A streptococcal infection, Group B streptococcalinfection, Haemophilus influenzae infection, Hand, foot and mouthdisease (HFMD), Hantavirus Pulmonary Syndrome, Helicobacter pyloriinfection, Hemolytic-uremic syndrome, Hemorrhagic fever with renalsyndrome, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, HepatitisE, Herpes simplex, Histoplasmosis, Hookworm infection, Human bocavirusinfection, Human ewingii ehrlichiosis, Human granulocytic anaplasmosis,Human metapneumovirus infection, Human monocytic ehrlichiosis, Humanpapillomavirus infection, Human parainfluenza virus infection,Hymenolepiasis, Epstein-Barr Virus Infectious Mononucleosis (Mono),Influenza, Isosporiasis, Kawasaki disease, Keratitis, Kingella kingaeinfection, Kuru, Lassa fever, Legionellosis (Legionnaires' disease),Legionellosis (Pontiac fever), Leishmaniasis, Leprosy, Leptospirosis,Listeriosis, Lyme disease (Lyme borreliosis), Lymphatic filariasis(Elephantiasis), Lymphocytic choriomeningitis, Malaria, Marburghemorrhagic fever, Measles, Melioidosis (Whitmore's disease),Meningitis, Meningococcal disease, Metagonimiasis, Microsporidiosis,Molluscum contagiosum, Mumps, Murine typhus (Endemic typhus), Mycoplasmapneumonia, Mycetoma, Myiasis, Neonatal conjunctivitis (Ophthalmianeonatorum), (New) Variant Creutzfeldt-Jakob disease (vCJD, nvCJD),Nocardiosis, Onchocerciasis (River blindness), Paracoccidioidomycosis(South American blastomycosis), Paragonimiasis, Pasteurellosis,Pediculosis capitis (Head lice), Pediculosis corporis (Body lice),Pediculosis pubis (Pubic lice, Crab lice), Pelvic inflammatory disease,Pertussis (Whooping cough), Plague, Pneumococcal infection, Pneumocystispneumonia, Pneumonia, Poliomyelitis, Prevotella infection, Primaryamoebic meningoencephalitis, Progressive multifocal leukoencephalopathy,Psittacosis, Q fever, Rabies, Rat-bite fever, Respiratory syncytialvirus infection, Rhinosporidiosis, Rhinovirus infection, Rickettsialinfection, Rickettsial-pox, Rift Valley fever, Rocky mountain spottedfever, Rotavirus infection, Rubella, Salmonellosis, SARS (Severe AcuteRespiratory Syndrome), Scabies, Schistosomiasis, Sepsis, Shigellosis(Bacillary dysentery), Shingles (Herpes zoster), Smallpox (Variola),Sporotrichosis, Staphylococcal food poisoning, Staphylococcal infection,Strongyloidiasis, Syphilis, Taeniasis, Tetanus (Lockjaw), Tinea barbae(Barber's itch), Tinea capitis (Ringworm of the Scalp), Tinea corporis(Ringworm of the Body), Tinea cruris (Jock itch), Tinea manuum (Ringwormof the Hand), Tinea nigra, Tinea pedis (Athlete's foot), Tinea unguium(Onychomycosis), Tinea versicolor (Pityriasis versicolor), Toxocariasis(Ocular Larva Migrans), Toxocariasis (Visceral Larva Migrans),Toxoplasmosis, Trichinellosis, Trichomoniasis, Trichuriasis (Whipworminfection), Tuberculosis, Tularemia, Ureaplasma urealyticum infection,Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Viralpneumonia, West Nile Fever, White piedra (Tinea blanca), Yersiniapseudotuber-culosis infection, Yersiniosis, Yellow fever, Zygomycosis.

The cell binding molecule, which is more preferred to be an antibodydescribed in this patent that are against pathogenic strains include,but are not limit, Acinetobacter baumannii, Actinomyces israelii,Actinomyces gerencseriae and Propionibacterium propionicus, Trypanosomabrucei, HIV (Human immunodeficiency virus), Entamoeba histolytica,Anaplasma genus, Bacillus anthracis, Arcanobacterium haemolyticum, Juninvirus, Ascaris lumbricoides, Aspergillus genus, Astroviridae family,Babesia genus, Bacillus cereus, multiple bacteria, Bacteroides genus,Balantidium coli, Baylisascaris genus, BK virus, Piedraia hortae,Blastocystis hominis, Blastomyces dermatitides, Machupo virus, Borreliagenus, Clostridium botulinum, Sabia, Brucella genus, usuallyBurkholderia cepacia and other Burkholderia species, Mycobacteriumulcerans, Caliciviridae family, Campylobacter genus, usually Candidaalbicans and other Candida species, Bartonella henselae, Group AStreptococcus and Staphylococcus, Trypanosoma cruzi, Haemophilusducreyi, Varicella zoster virus (VZV), Chlamydia trachomatis,Chlamydophila pneumoniae, Vibrio cholerae, Fonsecaea pedrosoi,Clonorchis sinensis, Clostridium difficile, Coccidioides immitis andCoccidioides posadasii, Colorado tick fever virus, rhinoviruses,coronaviruses, CJD prion, Crimean-Congo hemorrhagic fever virus,Cryptococcus neoformans, Cryptosporidium genus, Ancylostoma braziliense;multiple parasites, Cyclospora cayetanensis, Taenia solium,Cytomegalovirus, Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4)-Flaviviruses, Dientamoeba fragilis, Corynebacterium diphtheriae,Diphyllobothrium, Dracunculus medinensis, Ebolavirus, Echinococcusgenus, Ehrlichia genus, Enterobius vermicularis, Enterococcus genus,Enterovirus genus, Rickettsia prowazekii, Parvovirus B19, Humanherpesvirus 6 and Human herpesvirus 7, Fasciolopsis buski, Fasciolahepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily,Clostridium perfringens, Fusobacterium genus, Clostridium perfringens;other Clostridium species, Geotrichum candidum, GSS prion, Giardiaintestinalis, Burkholderia mallei, Gnathostoma spinigerum andGnathostoma hispidum, Neisseria gonorrhoeae, Klebsiella granulomatis,Streptococcus pyogenes, Streptococcus agalactiae, Haemophilusinfluenzae, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71,Sin Nombre virus, Helicobacter pylori, Escherichia coli O157:H7,Bunyaviridae family, Hepatitis A Virus, Hepatitis B Virus, Hepatitis CVirus, Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1,Herpes simplex virus 2, Histoplasma capsulatum, Ancylostoma duodenaleand Necator americanus, Hemophilus influenzae, Human bocavirus,Ehrlichia ewingii, Anaplasma phagocytophilum, Human metapneumovirus,Ehrlichia chaffeensis, Human papillomavirus, Human parainfluenzaviruses, Hymenolepis nana and Hymenolepis diminuta, Epstein-Barr Virus,Orthomy-xoviridae family, Isospora belli, Kingella kingae, Klebsiellapneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Kuru prion,Lassa virus, Legionella pneumophila, Legionella pneumophila, Leishmaniagenus, Mycobacterium leprae and Mycobacterium lepromatosis, Leptospiragenus, Listeria monocytogenes, Borrelia burgdorferi and other Borreliaspecies, Wuchereria bancrofti and Brugia malayi, Lymphocyticchoriomeningitis virus (LCMV), Plasmodium genus, Marburg virus, Measlesvirus, Burkholderia pseudomallei, Neisseria meningitides, Metagonimusyokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV),Mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, numerous speciesof bacteria (Actinomycetoma) and fungi (Eumycetoma), parasitic dipterousfly larvae, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prion,Nocardia asteroides and other Nocardia species, Onchocerca volvulus,Paracoccidioides brasiliensis, Paragonimus westermani and otherParagonimus species, Pasteurella genus, Pediculus humanus capitis,Pediculus humanus corporis, Phthirus pubis, Bordetella pertussis,Yersinia pestis, Streptococcus pneumoniae, Pneumocystis jirovecii,Poliovirus, Prevotella genus, Naegleria fowleri, JC virus, Chlamydophilapsittaci, Coxiella burnetii, Rabies virus, Streptobacillus moniliformisand Spirillum minus, Respiratory syncytial virus, Rhinosporidiumseeberi, Rhinovirus, Rickettsia genus, Rickettsia akari, Rift Valleyfever virus, Rickettsia rickettsii, Rotavirus, Rubella virus, Salmonellagenus, SARS coronavirus, Sarcoptes scabiei, Schistosoma genus, Shigellagenus, Varicella zoster virus, Variola major or Variola minor,Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus,Staphylococcus aureus, Streptococcus pyogenes, Strongyloidesstercoralis, Treponema pallidum, Taenia genus, Clostridium tetani,Trichophyton genus, Trichophyton tonsurans, Trichophyton genus,Epidermophyton floccosum, Trichophyton rubrum, and Trichophytonmentagrophytes, Trichophyton rubrum, Hortaea werneckii, Trichophytongenus, Malassezia genus, Toxocara canis or Toxocara cati, Toxoplasmagondii, Trichinella spiralis, Trichomonas vaginalis, Trichuristrichiura, Mycobacterium tuberculosis, Francisella tularensis,Ureaplasma urealyticum, Venezuelan equine encephalitis virus, Vibriocolerae, Guanarito virus, West Nile virus, Trichosporon beigelii,Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fevervirus, Mucorales order (Mucormycosis) and Entomophthorales order(Entomophthora-mycosis), Pseudomonas aeruginosa, Campylobacter (Vibrio)fetus, Aeromonas hydrophila, Edwardsiella tarda, Yersinia pestis,Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonellatyphimurium, Treponema pertenue, Treponema carateneum, Borreliavincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae,Pneumocystis carinii, Brucella abortus, Brucella suis, Brucellamelitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsiatsutsugumushi, Clamydia spp.; pathogenic fungi (Aspergillus fumigatus,Candida albicans, Histoplasma capsulatum); protozoa (Entomoebahistolytica, Trichomonas tenas, Trichomonas hominis, Tryoanosomagambiense, Trypanosoma rhodesiense, Leishmania donovani, Leishmaniatropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodiumvivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths(Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium,and hookworms).

Other conjugates in this invention for treatment of viral diseaseinclude, but are not limited to, antibodies against antigens ofpathogenic viruses, including as examples and not by limitation:Poxyiridae, Herpesviridae, Adenoviridae, Papovaviridae, Enteroviridae,Picomaviridae, Parvoviridae, Reoviridae, Retroviridae, influenzaviruses, parainfluenza viruses, mumps, measles, respiratory syncytialvirus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Non-A/Non-BHepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, Oncovirus[such as, HBV (Hepatocellular carcinoma), HPV (Cervical cancer, Analcancer), Kaposi's sarcoma-associated herpesvirus (Kaposi's sarcoma),Epstein-Barr virus (Nasopharyngeal carcinoma, Burkitt's lymphoma,Primary central nervous system lymphoma), MCPyV (Merkel cell cancer),SV40 (Simian virus 40), HCV (Hepatocellular carcinoma), HTLV-I (AdultT-cell leukemia/lymphoma)], Immune disorders caused virus: [such asHuman Immunodeficiency Virus (AIDS)]; Central nervous system virus:[such as, JCV (Progressive multifocal leukoencephalopathy), MeV(Subacute sclerosing panencephalitis), LCV (Lymphocyticchoriomeningitis), Arbovirus encephalitis, Orthomyxoviridae (probable)(Encephalitis lethargica), RV (Rabies), Chandipura virus, Herpesviralmeningitis, Ramsay Hunt syndrome type II; Poliovirus (Poliomyelitis,Post-polio syndrome), HTLV-I (Tropical spastic paraparesis)];Cytomegalovirus (Cytomegalovirus retinitis, HSV (Herpetic keratitis));Cardiovascular virus [such as CBV (Pericarditis, Myocarditis)];Respiratory system/acute viral nasopharyngitis/viral pneumonia:[Epstein-Barr virus (EBV infection/Infectious mononucleosis),Cytomegalovirus; SARS coronavirus (Severe acute respiratory syndrome)Orthomyxoviridae: Influenzavirus A/B/C (Influenza/Avian influenza),Paramyxovirus: Human parainfluenza viruses (Parainfluenza), RSV (Humanrespiratory syncytialvirus), hMPV]; Digestive system virus [MuV (Mumps),Cytomegalovirus (Cytomegalovirus esophagitis); Adenovirus (Adenovirusinfection); Rotavirus, Norovirus, Astrovirus, Coronavirus; HBV(Hepatitis B virus), CBV, HAV (Hepatitis A virus), HCV (Hepatitis Cvirus), HDV (Hepatitis D virus), HEV (Hepatitis E virus), HGV (HepatitisG virus)]; Urogenital virus [such as, BK virus, MuV (Mumps)].

According to a further object, the present invention also concernspharmaceutical compositions comprising the conjugate of the inventiontogether with a pharmaceutically acceptable carrier, diluent, orexcipient for treatment of cancers, infections or autoimmune disorders.The method for treatment of cancers, infections and autoimmune disorderscan be practiced in vitro, in vivo, or ex vivo. Examples of in vitrouses include treatments of cell cultures in order to kill all cellsexcept for desired variants that do not express the target antigen; orto kill variants that express undesired antigen. Examples of ex vivouses include treatments of hematopoietic stem cells (HSC) prior to theperformance of the transplantation (HSCT) into the same patient in orderto kill diseased or malignant cells. For instance, clinical ex vivotreatment to remove tumour cells or lymphoid cells from bone marrowprior to autologous transplantation in cancer treatment or in treatmentof autoimmune disease, or to remove T cells and other lymphoid cellsfrom allogeneic bone marrow or tissue prior to transplant in order toprevent graft-versus-host disease, can be carried out as follows. Bonemarrow is harvested from the patient or other individual and thenincubated in medium containing serum to which is added the conjugate ofthe invention, concentrations range from about 1 pM to 0.1 mM, for about30 minutes to about 48 hours at about 37° C. The exact conditions ofconcentration and time of incubation (=dose) are readily determined bythe skilled clinicians. After incubation, the bone marrow cells arewashed with medium containing serum and returned to the patient by i.v.infusion according to known methods. In circumstances where the patientreceives other treatment such as a course of ablative chemotherapy ortotal-body irradiation between the time of harvest of the marrow andreinfusion of the treated cells, the treated marrow cells are storedfrozen in liquid nitrogen using standard medical equipment.

CHEMOTHEROPEUTIC DRUGS/CYTOTOXIC AGENTS FOR SYNERGY

Chemotheropeutic drugs that can be used along with the present inventionfor synergy are small molecule drugs including cytotoxic agents. A“small molecule drug” is broadly used herein to refer to an organic,inorganic, or organometallic compound that may have a molecular weightof, for example, 100 to 2500, more suitably from 200 to 2000. Smallmolecule drugs are well characterized in the art, such as inWO05058367A2, and in U.S. Pat. No. 4,956,303, among others and areincorporated in their entirety by reference. The drugs include knowndrugs and those that may become known drugs.

Drugs that are known include, but not limited to,

1). Chemotherapeutic agents: a). Alkylating agents: such as Nitrogenmustards: chlorambucil, chlornaphazine, cyclophosphamide, dacarbazine,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, mannomustine, mitobronitol, melphalan, mitolactol,pipobroman, novembichin, phenesterine, prednimustine, thiotepa,trofosfamide, uracil mustard; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); Duocarmycin (includingthe synthetic analogues, KW-2189 and CBI-TMI); Benzodiazepine dimers(e.g., dimmers of pyrrolobenzodiazepine (PBD) or tomaymycin,indolinobenzodiazepines, imidazobenzothiadiazepines, oroxazolidinobenzodiazepines); Nitrosoureas: (carmustine, lomustine,chlorozotocin, fotemustine, nimustine, ranimustine); Alkylsulphonates:(busulfan, treosulfan, improsulfan and piposulfan); Triazenes:(dacarbazine); Platinum containing compounds: (carboplatin, cisplatin,oxaliplatin); aziridines, such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemel-amine, trietylenephosphoramide,triethylenethio-phosphaoramide and trimethylolomel-amine]; b). PlantAlkaloids: such as Vinca alkaloids: (vincristine, vinblastine,vindesine, vinorelbine, navelbin); Taxoids: (paclitaxel, docetaxol) andtheir analogs, Maytansinoids (DM1, DM2, DM3, DM4, maytansine andansamitocins) and their analogs, cryptophycins (particularlycryptophycin 1 and cryptophycin 8); epothilones, eleutherobin,discodermo-lide, bryostatins, dolostatins, auristatins, tubulysins,cephalostatins; pancratistatin; a sarcodictyin; spongistatin; c). DNATopoisomerase Inhibitors: such as [Epipodophyllins:(9-aminocamptothecin, camptothecin, crisnatol, daunomycin, etoposide,etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoicacids (retinols), teniposide, topotecan, 9-nitrocamptothecin (RFS2000)); mitomycins: (mitomycin C)]; d). Anti-metabolites: such as{[Anti-folate: DHFR inhibitors: (methotrexate, trimetrexate, denopterin,pteropterin, aminopterin (4-aminopteroic acid) or the other folic acidanalogues); IMP dehydrogenase Inhibitors: (mycophenolic acid,tiazofurin, ribavirin, EICAR); Ribonucleotide reductase Inhibitors:(hydroxyurea, deferoxamine)]; [Pyrimidine analogs: Uracil analogs:(ancitabine, azacitidine, 6-azauridine, capecitabine (Xeloda), carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-Fluorouracil,floxuridine, ratitrexed (Tomudex)); Cytosine analogs: (cytarabine,cytosine arabinoside, fludarabine); Purine analogs: (azathioprine,fludarabine, mercaptopurine, thiamiprine, thioguanine)]; folic acidreplenisher, such as frolinic acid}; e). Hormonal therapies: such as{Receptor antagonists: [Anti-estrogen: (megestrol, raloxifene,tamoxifen); LHRH agonists: (goscrclin, leuprolide acetate);Anti-androgens: (bicalutamide, flutamide, calusterone, dromostanolonepropionate, epitiostanol, goserelin, leuprolide, mepitiostane,nilutamide, testolactone, trilostane and other androgens inhibitors)];Retinoids/Deltoids: [Vitamin D3 analogs: (CB 1093, EB 1089 KH 1060,cholecalciferol, ergocalciferol); Photodynamic therapies: (verteporfin,phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A);Cytokines: (Interferon-alpha, Interferon-gamma, tumor necrosis factor(TNFs), human proteins containing a TNF domain)]}; f). Kinaseinhibitors, such as BIBW 2992 (anti-EGFR/Erb2), imatinib, gefitinib,pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib,lapatinib, axitinib, pazopanib. vandetanib, E7080 (anti-VEGFR2),mubritinib, ponatinib (AP24534), bafetinib (INNO—406), bosutinib(SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387,axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, Trastuzumab,Ranibizumab, Panitumumab, ispinesib; g). A poly (ADP-ribose) polymerase(PARP) inhibitors, such as olaparib, niraparib, iniparib, talazoparib,veliparib, veliparib, CEP 9722 (Cephalon's), E7016 (Eisai's), BGB-290(BeiGene's), 3-aminobenzamide.

h). antibiotics, such as the enediyne antibiotics (e.g. calicheamicins,especially calicheamicin γ1, δ1, α1 and β1, see, e.g., J. Med. Chem., 39(11), 2103-2117 (1996), Angew Chem Intl. Ed. Engl. 33:183-186 (1994);dynemicin, including dynemicin A and deoxydynemicin; esperamicin,kedarcidin, C-1027, maduropeptin, as well as neocarzinostatinchromophore and related chromoprotein enediyne antiobioticchromomophores), aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin;chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin,nitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin,potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; i). Others:such as Polyketides (acetogenins), especially bullatacin andbullatacinone; gemcitabine, epoxomicins (e.g. carfilzomib), bortezomib,thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032,STA-9090, Stimuvax, αllovectin-7, Xegeva, Provenge, Yervoy,Isoprenylation inhibitors (such as Lovastatin), Dopaminergic neurotoxins(such as 1-methyl-4-phenylpyridinium ion), Cell cycle inhibitors (suchas staurosporine), Actinomycins (such as Actinomycin D, dactinomycin),Bleomycins (such as bleomycin A2, bleomycin B2, peplomycin),Anthracyclines (such as daunorubicin, doxorubicin (adriamycin),idarubicin, epirubicin, pirarubicin, zorubicin, mtoxantrone, MDRinhibitors (such as verapamil), Ca²⁺ ATPase inhibitors (such asthapsigargin), Histone deacetylase inhibitors (Vorinostat, Romidepsin,Panobinostat, Valproic acid, Mocetinostat (MGCD0103), Belinostat,PCI-24781, Entinostat, SB939, Resminostat, Givinostat, AR-42, CUDC-101,sulforaphane, Trichostatin A); Thapsigargin, Celecoxib, glitazones,epigallocatechin gallate, Disulfiram, Salinosporamide A.; Anti-adrenals,such as aminoglutethimide, mitotane, trilostane; aceglatone;aldophosphamide glycoside; aminolevulinic acid; amsacrine; arabinoside,bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; eflornithine (DFMO), elfomithine; elliptinium acetate,etoglucid; gallium nitrate; gacytosine, hydroxyurea; ibandronate,lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine;pentostatin; phenamet; pirarubicin; podophyllinic acid;2-ethylhydrazide; procarbazine; PSK©; razoxane; rhizoxin; sizofiran;spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verrucarin A, roridin A and anguidine); urethane, siRNA, antisensedrugs, and a nucleolytic enzyme.

2). An anti-autoimmune disease agent includes, but is not limited to,cyclosporine, cyclosporine A, aminocaproic acid, azathioprine,bromocriptine, chlorambucil, chloroquine, cyclophosphamide,corticosteroids (e.g. amcinonide, betamethasone, budesonide,hydrocortisone, flunisolide, fluticasone propionate, fluocortolonedanazol, dexamethasone, Triamcinolone acetonide, beclometasonedipropionate), DHEA, enanercept, hydroxychloroquine, infliximab,meloxicam, methotrexate, mofetil, mycophenylate, prednisone, sirolimus,tacrolimus.

3). An anti-infectious disease agent includes, but is not limited to,a). Aminoglycosides: amikacin, astromicin, gentamicin (netilmicin,sisomicin, isepamicin), hygromycin B, kanamycin (amikacin, arbekacin,bekanamycin, dibekacin, tobramycin), neomycin (framycetin, paromomycin,ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin,verdamicin; b). Amphenicols:azidamfenicol, chloramphenicol, florfenicol,thiamphenicol; c). Ansamycins: geldanamycin, herbimycin; d).Carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin,meropenem, panipenem; e). Cephems: carbacephem (loracarbef),cefacetrile, cefaclor, cefradine, cefadroxil, cefalonium, cefaloridine,cefalotin or cefalothin, cefalexin, cefaloglycin, cefamandole,cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin,cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin,cefprozil, cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir,cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid,cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin,cefpimizole, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome,cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime,ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cephamycin (cefoxitin,cefotetan, cefmetazole), oxacephem (flomoxef, latamoxef); f).Glycopeptides: bleomycin, vancomycin (oritavancin, telavancin),teicoplanin (dalbavancin), ramoplanin; g). Glycylcyclines: e.g.tigecycline; g). β-Lactamase inhibitors: penam (sulbactam, tazobactam),clavam (clavulanic acid); i). Lincosamides: clindamycin, lincomycin; j).Lipopeptides: daptomycin, A54145, calcium-dependent antibiotics (CDA);k). Macrolides: azithromycin, cethromycin, clarithromycin,dirithromycin, erythromycin, flurithromycin, josamycin, ketolide(telithromycin, cethromycin), midecamycin, miocamycin, oleandomycin,rifamycins (rifampicin, rifampin, rifabutin, rifapentine), rokitamycin,roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506),troleandomycin, telithromycin; 1). Monobactams: aztreonam, tigemonam;m). Oxazolidinones: linezolid; n). Penicillins: amoxicillin, ampicillin(pivampicillin, hetacillin, bacampicillin, metampicillin,talampicillin), azidocillin, azlocillin, benzylpenicillin, benzathinebenzylpenicillin, benzathine phenoxymethyl-penicillin, clometocillin,procaine benzylpenicillin, carbenicillin (carindacillin), cloxacillin,dicloxacillin, epicillin, flucloxacillin, mecillinam (pivmecillinam),mezlocillin, meticillin, nafcillin, oxacillin, penamecillin, penicillin,pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin,sulbenicillin, temocillin, ticarcillin; o). Polypeptides: bacitracin,colistin, polymyxin B; p). Quinolones: alatrofloxacin, balofloxacin,ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin,enrofloxacin, floxin, garenoxacin, gatifloxacin, gemifloxacin,grepafloxacin, kano trovafloxacin, levofloxacin, lomefloxacin,marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin,ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin,sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q).Streptogramins: pristinamycin, quinupristin/dalfopristin); r).Sulfonamides: mafenide, prontosil, sulfacetamide, sulfamethizole,sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim,trimethoprim-sulfamethoxazole (co-trimoxazole); s). Steroidantibacterials: e.g. fusidic acid; t). Tetracyclines: doxycycline,chlortetracycline, clomocycline, demeclocycline, lymecycline,meclocycline, metacycline, minocycline, oxytetracycline,penimepicycline, rolitetracycline, tetracycline, glycylcyclines (e.g.tigecycline); u). Other types of antibiotics: annonacin, arsphenamine,bactoprenol inhibitors (Bacitracin), DADAL/AR inhibitors (cycloserine),dictyostatin, discodermolide, eleutherobin, epothilone, ethambutol,etoposide, faropenem, fusidic acid, furazolidone, isoniazid,laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesisinhibitors (e.g. fosfomycin), nitrofurantoin, paclitaxel, platensimycin,pyrazinamide, quinupristin/dalfopristin, rifampicin (rifampin),tazobactam tinidazole, uvaricin;

4). Anti-viral drugs: a). Entry/fusion inhibitors: aplaviroc, maraviroc,vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 (ibalizumab); b). Integraseinhibitors: raltegravir, elvitegravir, globoidnan A; c). Maturationinhibitors: bevirimat, vivecon; d). Neuraminidase inhibitors:oseltamivir, zanamivir, peramivir; e). Nucleosides & nucleotides:abacavir, aciclovir, adefovir, amdoxovir, apricitabine, brivudine,cidofovir, clevudine, dexelvucitabine, didanosine (ddI), elvucitabine,emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU),3′-fluoro-substituted 2′, 3′-dideoxynucleoside analogues (e.g.3′-fluoro-2′,3′-dideoxythymidine (FLT) and3′-fluoro-2′,3′-dideoxyguanosine (FLG), fomivirsen, ganciclovir,idoxuridine, lamivudine (3TC),1-nucleosides (e.g. β-1-thymidine andβ-1-2′-deoxycytidine), penciclovir, racivir, ribavirin, stampidine,stavudine (d4T), taribavirin (viramidine), telbivudine, tenofovir,trifluridine valaciclovir, valganciclovir, zalcitabine (ddC), zidovudine(AZT); f). Non-nucleosides: amantadine, ateviridine, capravirine,diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol,emivirine, efavirenz, foscarnet (phosphonoformic acid), imiquimod,interferon alfa, loviride, lodenosine, methisazone, nevirapine, NOV-205,peginterferon alfa, podophyllotoxin, rifampicin, rimantadine, resiquimod(R-848), tromantadine; g). Protease inhibitors: amprenavir,atazanavir,boceprevir, darunavir, fosamprenavir, indinavir, lopinavir,nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950),tipranavir; h). Other types of anti-virus drugs: abzyme, arbidol,calanolide a, ceragenin, cyanovirin-n, diarylpyrimidines,epigallocatechin gallate (EGCG), foscarnet, griffithsin, taribavirin(viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, portmanteauinhibitors, ribavirin, seliciclib.

5). The radioisotopes for radiotherapy. Examples of radioisotopes(radionuclides) are ³H, ¹¹C, ¹⁴C ¹⁸F, ³²P, ³⁵S ⁶⁴Cu, ⁶⁸Ga ⁸⁶Y⁹⁹Tc,¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³³Xe, ¹⁷⁷Lu, ²¹¹At, or ²¹³Bi.Radioisotope labeled antibodies are useful in receptor targeted imagingexperiments or can be for targeted treatment such as with theantibody-radioisotope conjugates (Wu et al (2005) Nature Biotechnology23(9): 1137-46). The cell binding molecules, e.g. an antibody can belabeled with ligand reagents that bind, chelate or otherwise complex aradioisotope metal, using the techniques described in Current Protocolsin Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience,New York, Pubs. (1991). Chelating ligands which may complex a metal ioninclude DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, Tex.USA).

6). Another cell-binding molecule-drug conjugate as a synergy therapy.The preferred synergic conjugate can be a conjugate having a cytotoxicagent of tubulysin analog, maytansinoid analog, taxanoid (taxane)analog, CC-1065 analog, daunorubicin and doxorubicin compound, amatoxinanalog, benzodiazepine dimer (e.g., dimers of pyrrolobenzodiazepine(PBD), tomaymycin, anthramycin, indolinobenzodiazepines,imidazobenzothiadiazepines, or oxazolidinobenzodiazepines),calicheamicins and the enediyne antibiotic compound, actinomycin,azaserine, bleomycins, epirubicin, tamoxifen, idarubicin, dolastatins,auristatins (e.g. monomethyl auristatin E, MMAE, MMAF, auristatin PYE,auristatin TP, Auristatins 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)),duocarmycins, geldanamycins, methotrexates, thiotepa, vindesines,vincristines, hemiasterlins, nazumamides, microginins, radiosumins,alterobactins, microsclerodermins, theonellamides, esperamicins,PNU-159682, and their analogues and derivatives above thereof.

7). The pharmaceutically acceptable salts, acids or derivatives of anyof the above drugs.

In yet another embodiment, an immunotoxin can be conjugated to acell-binding molecule as a synergic drug. An immunotoxin herein is amacromolecular drug which is usually a cytotoxic protein derived from abacterial or plant protein, such as Diphtheria toxin (DT), Cholera toxin(CT), Trichosanthin (TCS), Dianthin, Pseudomonas exotoxin A (ETA′),Erythrogenic toxins, Diphtheria toxin, AB toxins, Type III exotoxins,etc. It also can be a highly toxic bacterial pore-forming protoxin thatrequires proteolytic processing for activation. An example of thisprotoxin is proaerolysin and its genetically modified form, topsalysin.Topsalysin is a modified recombinant protein that has been engineered tobe selectively activated by an enzyme in the prostate, leading tolocalized cell death and tissue disruption without damaging neighboringtissue and nerves.

In another synergistic immunotherapy, an antibody of a checkpointinhibitor, TCR (T cell receptors) T cells, or CARs (chimeric antigenreceptors) T cells, or of B cell receptor (BCR), Natural killer (NK)cells, or the cytotoxic cells, or an antibody of anti-CD3, CD4, CD8,CD16 (FcγRIII), CD27, CD40, CD40L, CD45RA, CD45RO, CD56, CD57,CD57^(bright), TNFβ, Fas ligand, MHC class I molecules (HLA-A, B, C), orNKR-P1 is preferred to use along with the conjugates of the presentpatent for synergistic therapy.

Formulation and Application

The conjugates of the patent application are formulated to liquid, orsuitable to be lyophilized and subsequently be reconstituted to a liquidformulation. The conjugate in a liquid formula or in the formulatedlyophilized powder may take up 0.01%-99% by weight as major gradient inthe formulation. In general, a liquid formulation comprising 0.1 g/L−300g/L of concentration of the conjugate active ingredient for delivery toa patient without high levels of antibody aggregation may include one ormore polyols (e.g. sugars), a buffering agent with pH 4.5 to 7.5, asurfactant (e.g. polysorbate 20 or 80), an antioxidant (e.g. ascorbicacid and/or methionine), a tonicity agent (e.g. mannitol, sorbitol orNaCl), chelating agents such as EDTA; metal complexes (e.g. Zn-proteincomplexes); biodegradable polymers such as polyesters; a preservative(e.g. benzyl alcohol) and/or a free amino acid.

Suitable buffering agents for use in the formulations include, but arenot limited to, organic acid salts such as sodium, potassium, ammonium,or trihydroxyethylamino salts of citric acid, ascorbic acid, gluconicacid, carbonic acid, tartaric acid, succinic acid, acetic acid orphthalic acid; Tris, tromethamine hydrochloride, sulfate or phosphatebuffer. In addition, amino acid cationic components can also be used asbuffering agent. Such amino acid component includes without limitationarginine, glycine, glycylglycine, and histidine. The arginine buffersinclude arginine acetate, arginine chloride, arginine phosphate,arginine sulfate, arginine succinate, etc. In one embodiment, thearginine buffer is arginine acetate. Examples of histidine buffersinclude histidine chloride-arginine chloride, histidine acetate-arginineacetate, histidine phosphate-arginine phosphate, histidinesulfate-arginine sulfate, histidine succinate-argine succinate, etc. Theformulations of the buffers have a pH of 4.5 to pH 7.5, preferably fromabout 4.5 to about 6.5, more preferably from about 5.0 to about 6.2. Insome embodiments, the concentration of the organic acid salts in thebuffer is from about 10 mM to about 500 mM.

A “polyol” that may optionally be included in the formulation is asubstance with multiple hydroxyl groups. Polyols can be used asstabilizing excipients and/or isotonicity agents in both liquid andlyophilized formulations. Polyols can protect biopharmaceuticals fromboth physical and chemical degradation pathways. Preferentially excludedco-solvents increase the effective surface tension of solvent at theprotein interface whereby the most energetically favorable structuralconformations are those with the smallest surface areas. Polyols includesugars (reducing and nonreducing sugars), sugar alcohols and sugaracids. A “reducing sugar” is one which contains a hemiacetal group thatcan reduce metal ions or react covalently with lysine and other aminogroups in proteins and a “nonreducing sugar” is one which does not havethese properties of a reducing sugar. Examples of reducing sugars arefructose, mannose, maltose, lactose, arabinose, xylose, ribose,rhamnose, galactose and glucose. Nonreducing sugars include sucrose,trehalose, sorbose, melezitose and raffinose. Sugar alcohols areselected from mannitol, xylitol, erythritol, maltitol, lactitol,erythritol, threitol, sorbitol and glycerol. Sugar acids includeL-gluconate and metallic salts thereof. The polyol in the liquid formulaor in the formulated lyophilized solid can be 0.0%-20% by weight.Preferably, a nonreducing sugar, sucrose or trehalose at a concentrationof about from 0.1% to 15% is chosen in the formulation, whereintrehalose being preferred over sucrose, because of the solutionstability of trehalose.

A surfactant optionally in the formulations is selected from polysorbate(polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80,polysorbate 81, polysorbate 85 and the like); poloxamer (e.g. poloxamer188, poly(ethylene oxide)-poly(propylene oxide), poloxamer 407 orpolyethylene-polypropylene glycol and the like); Triton; sodium dodecylsulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-,myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-,linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamido-propyl-betaine(e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamido-propyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; dodecyl betaine, dodecyl dimethylamine oxide,cocamidopropyl betaine and coco ampho glycinate; and the MONAQUAT™series (e.g. isostearyl ethylimidonium ethosulfate); polyethyl glycol,polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g.Pluronics, PF68 etc); etc. Preferred surfactants are polyoxyethylenesorbitan fatty acid esters e.g. polysorbate 20, 40, 60 or 80 (Tween 20,40, 60 or 80). The concentration of a surfactant in the formulation isrange from 0.0% to about 2.0% by weight. In certain embodiments, thesurfactant concentration is from about 0.01% to about 0.2%. In oneembodiment, the surfactant concentration is about 0.02%.

A “preservative” optionally in the formulations is a compound thatessentially reduces bacterial action therein. Examples of potentialpreservatives include octadecyldimethylbenzyl ammonium chloride,hexamethonium chloride, benzalkonium chloride (a mixture ofalkylbenzyldimethylammonium chlorides in which the alkyl groups arelong-chain compounds), and benzethonium chloride. Other types ofpreservatives include aromatic alcohols such as phenol, butyl and benzylalcohol, alkyl parabens such as methyl or propyl paraben, catechol,resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The preservative inthe liquid formula or in the formulated lyophilized powder can be0.0%-5.0% by weight. In one embodiment, the preservative herein isbenzyl alcohol.

Suitable free amino acids as a bulky material, or tonicity agent, orosmotic pressure adjustment in the formulation, is selected from, butare not limited to, one or more of arginine, cystine, glycine, lysine,histidine, omithine, isoleucine, leucine, alanine, glycine glutamic acidor aspartic acid. The inclusion of a basic amino acid is preferred i.e.arginine, lysine and/or histidine. If a composition includes histidinethen this may act both as a buffering agent and a free amino acid, butwhen a histidine buffer is used it is typical to include a non-histidinefree amino acid e.g. to include histidine buffer and lysine. An aminoacid may be present in its D- and/or L-form, but the L-form is typical.The amino acid may be present as any suitable salt e.g. a hydrochloridesalt, such as arginine-HCl. The amino acid in the liquid formula or inthe formulated lyophilized powder can be 0.0%-30% by weight.

The formulations can optionally comprise methionine, glutathione,cysteine, cystine or ascorbic acid as an antioxidant at a concentrationof about up to 5 mg/ml in the liquid formula or 0.0%-5.0% by weight inthe formulated lyophilized powder; The formulations can optionallycomprise metal chelating agent, e.g., EDTA, EGTA, etc., at aconcentration of about up to 2 mM in the liquid formula or 0.0%-0.3% byweight in the formulated lyophilized powder.

The final formulation can be adjusted to the preferred pH with a bufferadjusting agent (e.g. an acid, such as HCl, H₂SO₄, acetic acid, H₃PO₄,citric acid, etc, or a base, such as NaOH, KOH, NH₄OH, ethanolamine,diethanolamine or triethanol amine, sodium phosphate, potassiumphosphate, trisodium citrate, tromethamine, etc) and the formulationshould be controlled “isotonic” which is meant that the formulation ofinterest has essentially the same osmotic pressure as human blood.Isotonic formulations will generally have an osmotic pressure from about250 to 350 mOsm. Isotonicity can be measured using a vapor pressure orice-freezing type osmometer, for example. The isotonic agent is selectedfrom mannitol, sorbitol, sodium acetate, potassium chloride, sodiumphosphate, potassium phosphate, trisodium citrate, or NaCl. In general,both the buffer salts and the isotonic agent may take up to 30% byweight in the formulation.

Other excipients which may be useful in either a liquid or lyophilizedformulation of the patent application include, for example, fucose,cellobiose, maltotriose, melibiose, octulose, ribose, xylitol, arginine,histidine, glycine, alanine, methionine, glutamic acid, lysine,imidazole, glycylglycine, mannosylglycerate, Triton X-100, PluoronicF-127, cellulose, cyclodextrin, (2-Hydroxypropyl)-β-cyclodextrin,dextran (10, 40 and/or 70 kD), polydextrose, maltodextrin, ficoll,gelatin, hydroxypropylmeth, sodium phosphate, potassium phosphate,ZnCl₂, zinc, zinc oxide, sodium citrate, trisodium citrate,tromethamine, copper, fibronectin, heparin, human serum albumin,protamine, glycerin, glycerol, EDTA, metacresol, benzyl alcohol, phenol,polyhydric alcohols, or polyalcohols, hydrogenated forms of carbohydratehaving a carbonyl group reduced to a primary or secondary hydroxylgroup.

Other contemplated excipients, which may be utilized in the aqueouspharmaceutical compositions of the patent application include, forexample, flavoring agents, antimicrobial agents, sweeteners,antioxidants, antistatic agents, lipids such as phospholipids or fattyacids, steroids such as cholesterol, protein excipients such as serumalbumin (human serum albumin), recombinant human albumin, gelatin,casein, salt-forming counterions such sodium and the like. These andadditional known pharmaceutical excipients and/or additives suitable foruse in the formulations of the invention are known in the art, e.g., aslisted in “The Handbook of Pharmaceutical Excipients, ₄1h edition, Roweet al., Eds., American Pharmaceuticals Association (2003); andRemington: the Science and Practice of Pharmacy, 21^(th) edition,Gennaro, Ed., Lippincott Williams & Wilkins (2005).

A pharmaceutical container or vessel is used to hold the pharmaceuticalformulation of any of conjugates of the patent application. The vesselis a vial, bottle, pre-filled syringe, pre-filled or auto-injectorsyringe. The liquid formula can be freeze-dried or drum-dried to a formof cake or powder in a borosilicate vial or soda lime glass vial. Thesolid powder can also be prepared by efficient spray drying, and thenpacked to a vial or a pharmaceutical container for storage anddistribution.

In a further embodiment, the invention provides a method for preparing aformulation comprising the steps of: (a) lyophilizing the formulationcomprising the conjugates, excipients, and a buffer system; and (b)reconstituting the lyophilized mixture of step (a) in a reconstitutionmedium such that the reconstituted formulation is stable. Theformulation of step (a) may further comprise a stabilizer and one ormore excipients selected from a group comprising bulking agent, salt,surfactant and preservative as hereinabove described. As reconstitutionmedia, several diluted organic acids or water, i.e. sterile water,bacteriostatic water for injection (BWFI) or may be used. Thereconstitution medium may be selected from water, i.e. sterile water,bacteriostatic water for injection (BWFI) or the group consisting ofacetic acid, propionic acid, succinic acid, sodium chloride, magnesiumchloride, acidic solution of sodium chloride, acidic solution ofmagnesium chloride and acidic solution of arginine, in an amount fromabout 10 to about 250 mM.

A liquid pharmaceutical formulation of the conjugates of the patentapplication should exhibit a variety of pre-defined characteristics. Oneof the major concerns in liquid drug products is stability, asproteins/antibodies tend to form soluble and insoluble aggregates duringmanufacturing and storage. In addition, various chemical reactions canoccur in solution (deamination, oxidation, clipping, isomerization etc.)leading to an increase in degradation product levels and/or loss ofbioactivity. Preferably, a conjugate in either liquid or lyophilizationformulation should exhibit a shelf life of more than 6 months at 25° C.More preferred a conjugate in either liquid or lyophilizationformulation should exhibit a shelf life of more than 12 months at 25° C.Most preferred liquid formulation should exhibit a shelf life of about24 to 36 months at 2-8° C. and the lyophilization formulation shouldexhibit a shelf life of about preferably up to 60 months at 2-8° C. Bothliquid and lyophilization formulations should exhibit a shelf life forat least two years at −20° C., or −70° C.

In certain embodiments, the formulation is stable following freezing(e.g., −20° C., or −70° C.) and thawing of the formulation, for examplefollowing 1, 2 or 3 cycles of freezing and thawing. Stability can beevaluated qualitatively and/or quantitatively in a variety of differentways, including evaluation of drug/antibody(protein) ratio and aggregateformation (for example using UV, size exclusion chromatography, bymeasuring turbidity, and/or by visual inspection); by assessing chargeheterogeneity using cation exchange chromatography, image capillaryisoelectric focusing (icIEF) or capillary zone electrophoresis;amino-terminal or carboxy-terminal sequence analysis; mass spectrometricanalysis, or matrix-assisted laser desorption ionization/time-of-flightmass spectrometry (MALDI/TOF MS), or HPLC-MS/MS; SDS-PAGE analysis tocompare reduced and intact antibody; peptide map (for example tryptic orLYS--C) analysis; evaluating biological activity or antigen bindingfunction of the antibody; etc. Instability may involve any one or moreof: aggregation, deamination (e.g. Asn deamination), oxidation (e.g. Metoxidation), isomerization (e.g. Asp isomerization),clipping/hydrolysis/fragmentation (e.g. hinge region fragmentation),succinimide formation, unpaired cysteine(s), N-terminal extension,C-terminal processing, glycosylation differences, etc.

A stable conjugate should also “retains its biological activity” in apharmaceutical formulation, if the biological activity of the conjugateat a given time, e.g. 12 month, within about 20%, preferably about 10%(within the errors of the assay) of the biological activity exhibited atthe time the pharmaceutical formulation was prepared as determined in anantigen binding assay, and/or in vitro, cytotoxic assay, for example.

For clinical in vivo use, the conjugate via the bis-linkage of theinvention will be supplied as solutions or as a lyophilized solid thatcan be redissolved in sterile water for injection. Examples of suitableprotocols of conjugate administration are as follows. Conjugates aregiven daily, weekly, biweekly, triweekly, once every four weeks ormonthly for 8-54 weeks as an i.v. bolus. Bolus doses are given in 50 to1000 ml of normal saline to which human serum albumin (e.g. 0.5 to 1 mLof a concentrated solution of human serum albumin, 100 mg/mL) canoptionally be added. Dosages will be about 50 μg to 20 mg/kg of bodyweight per week, i.v. (range of 10 μg to 200 mg/kg per injection). 4-54weeks after treatment, the patient may receive a second course oftreatment. Specific clinical protocols with regard to route ofadministration, excipients, diluents, dosages, times, etc., can bedetermined by the skilled clinicians.

Examples of medical conditions that can be treated according to the invivo or ex vivo methods of killing selected cell populations includemalignancy of any types of cancer, autoimmune diseases, graftrejections, and infections (viral, bacterial or parasite).

The amount of a conjugate which is required to achieve the desiredbiological effect, will vary depending upon a number of factors,including the chemical characteristics, the potency, and thebioavailability of the conjugates, the type of disease, the species towhich the patient belongs, the diseased state of the patient, the routeof administration, all factors which dictate the required dose amounts,delivery and regimen to be administered.

In general terms, the conjugates via the bis-linkers of this inventionmay be provided in an aqueous physiological buffer solution containing0.1 to 10% w/v conjugates for parenteral administration. Typical doseranges are from 1 μg/kg to 0.1 g/kg of body weight daily; weekly,biweekly, triweekly, or monthly, a preferred dose range is from 0.01mg/kg to 20 mg/kg of body weight weekly, biweekly, triweekly, ormonthly, an equivalent dose in a human. The preferred dosage of drug tobe administered is likely to depend on such variables as the type andextent of progression of the disease or disorder, the overall healthstatus of the particular patient, the relative biological efficacy ofthe compound selected, the formulation of the compound, the route ofadministration (intravenous, intramuscular, or other), thepharmacokinetic properties of the conjugates by the chosen deliveryroute, and the speed (bolus or continuous infusion) and schedule ofadministrations (number of repetitions in a given period of time).

The conjugates via the linkers of the present invention are also capableof being administered in unit dose forms, wherein the term “unit dose”means a single dose which is capable of being administered to a patient,and which can be readily handled and packaged, remaining as a physicallyand chemically stable unit dose comprising either the active conjugateitself, or as a pharmaceutically acceptable composition, as describedhereinafter. As such, typical total daily/weekly/biweekly/monthly doseranges are from 0.01 to 100 mg/kg of body weight. By way of generalguidance, unit doses for humans range from 1 mg to 3000 mg per day, orper week, per two weeks (biweekly), triweekly, or per month. Preferably,the unit dose range is from 1 to 500 mg administered one to four times amonth and even more preferably from 1 mg to 100 mg, once a week,biweekly, or triweekly. Conjugates provided herein can be formulatedinto pharmaceutical compositions by admixture with one or morepharmaceutically acceptable excipients. Such unit dose compositions maybe prepared for use by oral administration, particularly in the form oftablets, simple capsules or soft gel capsules; or intranasal,particularly in the form of powders, nasal drops, or aerosols; ordermally, for example, topically in ointments, creams, lotions, gels orsprays, or via transdermal patches.

In yet another embodiment, a pharmaceutical composition comprising atherapeutically effective amount of the conjugate of Formula (I) orFormula (III) or any conjugates described through the present patent canbe administered concurrently with the other therapeutic agents such asthe chemotherapeutic agent, the radiation therapy, immunotherapy agents,autoimmune disorder agents, anti-infectious agents or the otherconjugates for synergistically effective treatment or prevention of acancer, or an autoimmune disease, or an infectious disease. Thesynergistic agents are preferably selected from one or several of thefollowing drugs: Abatacept, abemaciclib, Abiraterone acetate, Abraxane,Acetaminophen/hydrocodone, Acalabrutinib, aducanumab, Adalimumab,ADXS31-142, ADXS-HER2, afatinib dimaleate, aldesleukin, alectinib,alemtuzumab, Alitretinoin, ado-trastuzumab emtansine,Amphetamine/dextroamphetamine, anastrozole, Aripiprazole,anthracyclines, Aripiprazole, Atazanavir, Atezolizumab, Atorvastatin,Avelumab, Axicabtagene ciloleucel, axitinib, belinostat, BCG Live,Bevacizumab, bexarotene, blinatumomab, Bortezomib, bosutinib,brentuximab vedotin, brigatinib, Budesonide, Budesonide/formoterol,Buprenorphine, Cabazitaxel, Cabozantinib, capmatinib, Capecitabine,carfilzomib, chimeric antigen receptor-engineered T (CAR-T) cells,Celecoxib, ceritinib, Cetuximab, Chidamide, Ciclosporin, Cinacalcet,crizotinib, Cobimetinib, Cosentyx, crizotinib, CTL019, Dabigatran,dabrafenib, dacarbazine, daclizumab, dacomotinib, daptomycin,Daratumumab, Darbepoetin alfa, Darunavir, dasatinib, denileukindiftitox, Denosumab, Depakote, Dexlansoprazole, Dexmethylphenidate,Dexamethasone, DigniCap Cooling System, Dinutuximab, Doxycycline,Duloxetine, Duvelisib, durvalumab, elotuzumab,Emtricibine/Rilpivirine/Tenofovir, disoproxil fumarate,Emtricitbine/tenofovir/efavirenz, Enoxaparin, ensartinib, Enzalutamide,Epoetin alfa, erlotinib, Esomeprazole, Eszopiclone, Etanercept,Everolimus, exemestane, everolimus, exenatide ER, Ezetimibe,Ezetimibe/simvastatin, Fenofibrate, Filgrastim, fingolimod, Fluticasonepropionate, Fluticasone/salmeterol, fulvestrant, gazyva, gefitinib,Glatiramer, Goserelin acetate, Icotinib, Imatinib, Ibritumomab tiuxetan,ibrutinib, idelalisib, ifosfamide, Infliximab, imiquimod, ImmuCyst,Immuno BCG, iniparib, Insulin aspart, Insulin detemir, Insulin glargine,Insulin lispro, Interferon alfa, Interferon alfa-1b, Interferon alfa-2a,Interferon alfa-2b, Interferon beta, Interferon beta Ta, Interferon betaTb, Interferon gamma-Ta, lapatinib, Ipilimumab, Ipratropiumbromide/salbutamol, Ixazomib, Kanuma, Lanreotide acetate, lenalidomide,lenaliomide, lenvatinib mesylate, letrozole, Levothyroxine,Levothyroxine, Lidocaine, Linezolid, Liraglutide, Lisdexamfetamine,LN-144, lorlatinib, Memantine, Methylphenidate, Metoprolol, Mekinist,mericitabine/Rilpivirine/Tenofovir, Modafinil, Mometasone, Mycidac-C,Necitumumab, neratinib, Nilotinib, niraparib, Nivolumab, ofatumumab,obinutuzumab, olaparib, Olmesartan, Olmesartan/hydrochlorothiazide,Omalizumab, Omega-3 fatty acid ethyl esters, Oncorine, Oseltamivir,Osimertinib, Oxycodone, palbociclib, Palivizumab, panitumumab,panobinostat, pazopanib, pembrolizumab, PD-1 antibody, PD-L1 antibody,Pemetrexed, pertuzumab, Pneumococcal conjugate vaccine, pomalidomide,Pregabalin, ProscaVax, Propranolol, Quetiapine, Rabeprazole, radium 223chloride, Raloxifene, Raltegravir, ramucirumab, Ranibizumab,regorafenib, ribociclib, Rituximab, Rivaroxaban, romidepsin,Rosuvastatin, ruxolitinib phosphate, Salbutamol, savolitinib,semaglutide, Sevelamer, Sildenafil, siltuximab, Sipuleucel-T,Sitagliptin, Sitagliptin/metformin, Solifenacin, solanezumab, Sonidegib,Sorafenib, Sunitinib, tacrolimus, tacrimus, Tadalafil, tamoxifen,Tafinlar, Talimogene laherparepvec, talazoparib, Telaprevir,talazoparib, Temozolomide, temsirolimus, Tenofovir/emtricitabine,tenofovir disoproxil fumarate, Testosterone gel, Thalidomide, TICE BCG,Tiotropium bromide, Tisagenlecleucel, toremifene, trametinib,Trastuzumab, Trabectedin (ecteinascidin 743), trametinib, tremelimumab,Trifluridine/tipiracil, Tretinoin, Uro-BCG, Ustekinumab, Valsartan,veliparib, vandetanib, vemurafenib, venetoclax, vorinostat,ziv-aflibercept, Zostavax, and their analogs, derivatives,pharmaceutically acceptable salts, carriers, diluents, or excipientsthereof, or a combination above thereof.

The drugs/cytotoxic agents used for conjugation via a branched linker ofthe present patent can be any analogues and/or derivatives of tubulysindescribed in the present patent. One skilled in the art ofdrugs/cytotoxic agents will readily understand that each of thetubulysin described herein can be modified in such a manner that theresulting compound still retains the specificity and/or activity of thestarting compound. The skilled artisan will also understand that many ofthese analog or derivative compounds can be used in place of thetubulysin analogs described herein.

Thus, the tubulysin analogs of the present invention include manyanalogues and derivatives of the tubulysin compounds that may not bedescribed in detail thereof.

All references cited herein and in the examples that follow areexpressly incorporated by reference in their entireties.

EXAMPLES

The invention is further described in the following examples, which arenot intended to limit the scope of the invention. Cell lines describedin the following examples were maintained in culture according to theconditions specified by the American Type Culture Collection (ATCC) orDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Braunschweig, Germany (DMSZ), or The Shanghai Cell Culture Institute ofChinese Acadmy of Science, unless otherwise specified. Cell culturereagents were obtained from Invitrogen Corp., unless otherwisespecified. All anhydrous solvents were commercially obtained and storedin Sure-seal bottles under nitrogen. All other reagents and solventswere purchased as the highest grade available and used without furtherpurification. The preparative HPLC separations were performed withVarain PreStar HPLC. NMR spectra were recorded on Bruker 500 MHzInstrument. Chemical shifts (delta) are reported in parts per million(ppm) referenced to tetramethylsilane at 0.00 and coupling constants (J)are reported in Hz. The mass spectral data were acquired on a WatersXevo QTOF mass spectrum equipped with Waters Acquity UPLC separationsmodule and Acquity TUV detector.

Example 1. Synthesis of di-tert-butyl1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate

To di-tert-butyl hydrazine-1,2-dicarboxylate (8.01 g, 34.4 mmol) in DMF(150 ml) was added NaH (60% in oil, 2.76 g, 68.8 mmol). After stirred atRT for 30 min, tert-butyl 2-bromoacetate (14.01 g, 72.1 mmol) was added.The mixture was stirred overnight, quenched with addition of methanol (3ml), concentrated, diluted with EtOAc (100 ml) and water (100 ml),separated, and the aqueous layer was extracted with EtOAc (2×50 ml). Theorganic layers were combined, dried over MgSO₄, filtered, evaporated,and purified by SiO₂ column chromatography (EtOAc/Hexanel:5 to 1:3) toafforded the title compound (12.98 g, 82% yield) as a colorless oil.MSESI m/z calcd for C₂₂H₄₁N₂O₈ [M+H]⁺ 461.28, found 461.40.

Example 2. Synthesis of 2,2′-(hydrazine-1,2-diyl)diacetic acid

Di-tert-butyl1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate (6.51 g,14.14 mmol) in 1,4-dioxane (40 ml) was added HCl (12 M, 10 ml). Themixture was stirred for 30 min, diluted with dioxane (20 ml) and toluene(40 ml), evaporated and co-evaporated with dioxane (20 ml) and toluene(40 ml) to dryness to afford the crude title product for the next stepwithout further production (2.15 g, 103% yield, ˜93% pure). MS ESI m/zcalcd for C₄H₉N₂O₄ [M+H]⁺ 149.05, found 149.40.

Example 3. Synthesis of2,2′-(1,2-bis((E)-3-bromoacryloyl)hydrazine-1,2-diyl)diacetic acid

To a solution of 2,2′-(hydrazine-1,2-diyl)diacetic acid (1.10 g, 7.43mmol) in the mixture of THF (50 ml) and NaH₂PO₄ (0.1 M, 80 ml, pH 6.0)was added(E)-3-bromoacryloyl bromide (5.01 g, 23.60 mmol). The mixturewas stirred for 6 h, concentrated and purified on SiO₂ column elutedwith H₂O/CH₃CN (1:9) containing 3% formic acid to afford the titlecompound (2.35 g, 77% yield, ˜93% pure). MS ESI m/z calcd forC₁₀H₁₁Br₂N₂O₆ [M+H]⁺ 412.89, found 413.50.

Example 4. Synthesis of2,2′-(1,2-bis((E)-3-bromoacryloyl)hydrazine-1,2-diyl)diacetyl chloride

2,2′-(1,2-Bis((E)-3-bromoacryloyl)hydrazine-1,2-diyl)diacetic acid (210mg, 0.509 mmol) in dichloroethane (15 ml) was added (COCl)₂ (505 mg,4.01 mmol), followed by addition of 0.040 ml of DMF. After stirred at RTfor 2 h, the mixture was concentrated and co-evaporated withdichloroethane (2×20 ml) and toluene (2×15 ml) to dryness to affordedthe title crude product (which is not stable) for the next step withoutfurther purification (245 mg, 107% yield). MS ESI m/z calcd forC₁₀H₉Br₂Cl₂N₂O₄ [M+H]⁺ 448.82, 450.82, 452.82, 454.82, found 448.60,450.60, 452.60, 454.60.

Example 5. Synthesis of tert-butyl 2,8-dioxo-1,5-oxazocane-5-carboxylate

To a solution of 3,3′-azanediyldipropanoic acid(10.00 g, 62.08 mmol) in1.0 M NaOH (300 ml) at 4° C. was added di-tert-butyl dicarbonate (22.10g, 101.3 mmol) in 200 ml THF in 1 h. After addition, the mixture waskept to stirring for 2 h at 4° C. The mixture was carefully acidified topH ˜4 with 0.2 M H₃PO₄, concentrated in vacuo, extracted with CH₂Cl₂,dried over Na₂SO₄, evaporated and purified with flash SiO₂chromatography eluted with AcOH/MeOH/CH₂Cl₂ (0.01:1:5) to afford3,3′-((tert-butoxycarbonyl)azanediyl)dipropanoic acid(13.62 g, 84%yield).ESI MS m/z C₁₁H₁₉NO₆ [M+H]⁺, cacld. 262.27, found 262.40.

To a solution of 3,3′-((tert-butoxycarbonyl)azanediyl)dipropanoic acid(8.0 g, 30.6 mmol) in CH₂Cl₂ (500 ml) at 0° C. was added phosphoruspentoxide (8.70 g, 61.30 mmol). The mixture was stirred at 0° C. for 2 hand then r.t. for 1 h, filtered through short SiO₂ column, and rinsedthe column with EtOAc/CH₂Cl₂ (1:6). The filtrate was concentrated andtriturated with EtOAc/hexane to afford the title compound(5.64 g, 74%yield). ESI MS m/z C₁₁H₁₇NO₅ [M+H]⁺, cacld. 244.11, found 244.30.

Example 6. Synthesis of 2,5-dioxopyrrolidin-1-yl propiolate

Propiolic acid(5.00 g, 71.4 mmol), NHS (9.01 g, 78.3 mmol) and EDC (20.0g, 104.1 mmol) in CH₂Cl₂ (150 ml) and DIPEA (5 ml, 28.7 mmol) wasstirred for overnight, evaporated and purified by SiO₂ columnchromatography (EtOAc/Hexanel:4) to afforded the title compound (9.30 g,79% yield) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 2.68 (s, 1H),2.61 (s, 4H). MS ESI m/z calcd for C₇H₅NaNO₄ [M+Na]⁺190.02, found190.20.

Example 7. Synthesis of tert-butyl 2-propioloylhydrazinecarboxylate

Propiolic acid(5.00 g, 71.4 mmol), tert-butyl hydrazinecarboxylate (9.45g, 71.5 mmol) and EDC (20.0 g, 104.1 mmol) in CH₂Cl₂ (150 ml) and DIPEA(5 ml, 28.7 mmol) was stirred for overnight, evaporated and purified bySiO₂ column chromatography (EtOAc/Hexanel:5) to afforded the titlecompound (7.92 g, 84% yield) as a colorless oil. ¹H NMR (500 MHz, CDCl₃)δ 8.76 (m, 2H), 2.68 (s, 1H), 1.39 (s, 9H). MS ESI m/z calcd forC₅H₁₂NaN₂O₂[M+Na]⁺155.09, found 155.26.

Example 8. Synthesis of Propiolohydrazide, HCl Salt

tert-butyl 2-propioloylhydrazinecarboxylate(4.01 g, 30.35 mmol)dissolved in 1,4-dioxane (12 mL) was treated with 4 ml of HCl (conc.) at4° C. The mixture was stirred for 30 min, diluted with Dioxane (30 ml)and toluene (30 ml) and concentrated under vacuum. The crude mixture waspurified on silica gel using a mixture of methanol (from 5% to 10%) and1% formic acid in methylene chloride as the eluant to give titlecompound (2.11 g, 83% yield), ESI MS m/z C₃H₅N₂O [M+H]⁺, cacld. 85.03,found 85.30.

Example 9. Synthesis of Compound 2

In a 10-L reactor 2,2-diethoxyacetonitrile (1.00 kg, 7.74 mol, 1.0 eq.)was mixed with (NH₄)₂S (48% aqueous solution, 1.41 kg, 9.29 mol, 1.2eq.) in methanol (6.0 L) at room temperature. The internal temperatureincreased to 33° C. and then dropped back to r.t. After stirringovernight, the reaction mixture was concentrated under vacuum and theresidue was taken up in ethyl acetate (5 L) and washed with saturatedNaHCO₃ solution (4×1.0 L). The aqueous layer was back-extracted withethyl acetate (5×1.0 L). The organic phases were combined and washedwith brine (3 L), dried over anhydrous Na₂SO₄ and concentrated. Theresulting solid was collected by vacuum filtration and washed withpetroleum ether. The filtrate was concentrated and triturated withpetroleum ether to yield a few crops of white or light yellow solid. Allcrops were combined to give 1.1 kg of desired product (87% yield). ¹HNMR (500 MHz, CDCl₃) δ 7.81 (d, J=71.1 Hz, 2H), 5.03 (s, 1H), 3.73 (dq,J=9.4, 7.1 Hz, 2H), 3.64 (dq, J=9.4, 7.0 Hz, 2H), 1.25 (t, J=7.1 Hz,6H).

Example 10. Synthesis of Compound 3

In a 5-L3-neck round bottle flask, equipped with a reflux condenser andan additional funnel, ethyl bromopyruvate (80% purity, 404 mL, 2.57 mol,1.2 eq.) was added over 30 min. to a mixture of molecular sieves (3 Å,500 g) and thioamide(350 g, 2.14 mol, 1.0 eq.) in 3 L EtOH.

During addition, the internal temperature increased slightly. Thereaction mixture was then heated to reflux and stirred for 30 min. Aftercooling to r.t. the reaction mixture was filter over Celite and thefilter cake washed with ethyl acetate. The filtrate was concentratedunder vacuum. Two batches of the crude product were combined and mixedwith silica gel (1.5 kg) and loaded on a silica gel (10 kg packed)column and eluted with ethyl acetate/petroleum ether (10-20%) to givethiazole carboxylate as a brown oil (509 g, 92% yield).

Example 11. Synthesis of Compound 4

A solution of acetal(300 g, 1.16 mol) in acetone (3.0 L) was heated toreflux and 4N HCl (250 mL) was added over 1.0 h to the refluxingsolution. TLC analysis indicated complete consumption of the startingmaterial. The reaction mixture was concentrated under reduced pressureand phases were separated. The organic phase was diluted with ethylacetate (1.5 L) and washed with saturated NaHCO₃ solution (1.0 L), water(1.0 L) and brine (1.0 L), and then dried over anhydrous Na₂SO₄. All ofthe aqueous phases were combined and extracted with ethyl acetate. Theextracts were combined and dried over anhydrous Na₂SO₄. The organicsolutions were filtered and concentrated under reduced pressure. Thecrude product was triturated with petreolum ether and diethyl ether(5:1) and the resulting solid was collected by vacuum filtration andwashed with petreolum ether and ethyl acetate (10:1). The filtrate wasconcentrated and chromatographed using 0-15% ethyl acetate/petreolumether to give another crop of desired product. All white to light yellowsolids were combined and weighed 40 g (43% yield). ¹H NMR (500 MHz,CDCl₃) δ 10.08-10.06 (m, 1H), 8.53-8.50 (m, 1H), 4.49 (q, J=7.1 Hz, 2H),1.44 (t, J=7.1 Hz, 3H). MS ESI m/z calcd for C₇HSNO₃S [M+H]⁺ 186.01;found 186.01.

Example 12. Synthesis of Compound 6

NaN₃ (740 g, 11.4 mol) was dissolved in water (2.0 L) anddichloromethane (2.0 L) was added and cooled at 0° C., to which Tf₂O(700mL, 4.10 mol, 1.8 eq.) was added over 1.5 h. After addition wascompleted, the reaction was stirred at 0° C. for 3 h. The organic phasewas separated and the aqueous phase was extracted with dichloromethane(2×500 mL). The combined organic phases were washed with saturatedNaHCO₃ solution (3×1.0 L). This dichloromethane solution of triflylazide was added to a mixture of (L)-isoleucine (300 g, 2.28 mol, 1.0eq.), K₂CO₃ (472 g, 3.42 mol, 1.5 eq.), CuSO₄·5H₂O (5.7 g, 22.8 mmol,0.01 eq.) in water (3.0 L) and methanol (3.0 L) at r.t. During addition,the internal temperature increased slightly. And the mixture was thenstirred at r.t. for 16 h. The organic solvents were removed underreduced pressure and the aqueous phase was acidified to pH 6-6.5 withconcentrated HCl (about 280 mL added) and then diluted with phosphatebuffer (0.25 M, pH 6.2, 6.0 L), washed with EtOAc (6×2.0 L) to removethe sulfonamide by-product. The solution was acidified to pH 3 withconcentrated HCl (about 400 mL added), extracted with EtOAc (4×2.0 L).The combined organic layers were washed with brine (2.0 L) and driedover anhydrous Na₂SO₄, filtered and concentrated to give product 6 (320g, 89% yield) as a light yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 12.01 (s,1H), 3.82 (d, J=5.9 Hz, 1H), 2.00 (ddd, J=10.6, 8.6, 5.5 Hz, 1H), 1.54(dqd, J=14.8, 7.5, 4.4 Hz, 1H), 1.36-1.24 (m, 1H), 1.08-0.99 (m, 3H),0.97-0.87 (m, 3H).

Example 13. Synthesis of Compound 10

To a solution of (S)—2-methylpropane-2-sulfinamide (100 g, 0.825 mol,1.0 eq.) in 1 L THF was added Ti(OEt)₄ (345 mL, 1.82 mol, 2.2 eq.) and3-methyl-2-butanone (81 mL, 0.825 mol, 1.0 eq.) under N₂ at r.t. Thereaction mixture was refluxed for 16 h, then cooled to r.t. and pouredonto iced water (1 L). The mixture was filtered and the filter cake waswashed with EtOAc. The organic layer was separated, dried over anhydrousNa₂SO₄ and concentrated to give a residue which was purified by vacuumdistillation (15-20 torr, 95° C.) to afforded product 10 (141 g, 90%yield) as a yellow oil. 1H NMR (500 MHz, CDCl₃) δ 2.54-2.44 (m, 1H),2.25 (s, 3H), 1.17 (s, 9H), 1.06 (dd, J=6.9, 5.1 Hz, 6H). MS ESI m/zcalcd for C₉H₁₉NaNOS [M+Na]⁺212.12; found 212.11.

Example 14. Synthesis of Compound 11

To a solution of diisopropylamine (264 mL, 1.87 mol, 1.65 eq.) in dryTHF (1 L) was added n-butyllithium (2.5 M, 681 mL, 1.70 mol, 1.5 eq.) at−78° C. under N₂. The reaction mixture was warmed to 0° C. over 30 minand then cooled back to −78°. Compound 10 (258 g, 1.36 mol, 1.2 eq.) wasadded, and rinsed with THF (50 mL). The reaction mixture was stirred for1 h before CITi(O_(j)Pr)₃ (834 g, 3.17 mol, 2.8 eq.) in THF (1.05 L) wasadded dropwise. After stirring for 1 h, compound 4 (210 g, 1.13 mol, 1.0eq.) dissolved in THF (500 mL) was added dropwise in about 1 hours andthe resulting reaction mixture was stirred for 3 h. The completion ofthe reaction was indicated by TLC analysis. The reaction was quenched bya mixture of acetic acid and THF (v/v 1:1, 300 mL), then poured ontobrine (2 L), extracted with EtOAc (8×1 L). The organic phase was washedwith water and brine, dried over anhydrous Na₂SO₄, filtered andconcentrated. The residue was purified by column chromatography(DCM/EtOAc/PE 2:1:2) to afforded the compound 11 (298 g, 74% yield) as acolorless oil. ¹H NMR (500 MHz, CDCl₃) δ 8.13 (s, 1H), 6.63 (d, J=8.2Hz, 1H), 5.20-5.11 (m, 1H), 4.43 (q, J=7.0 Hz, 2H), 3.42-3.28 (m, 2H),2.89 (dt, J=13.1, 6.5 Hz, 1H), 1.42 (t, J=7.1 Hz, 3H), 1.33 (s, 9H),1.25-1.22 (m, 6H). MS ESI m/z calcd for C₁₆H₂₆NaN₂O₄S₂ [M+Na]⁺397.13,found 397.11.

Example 15. Synthesis of Compound 12

A solution of compound 11 (509 g, 1.35 mol, 1.0 eq.) dissolved in THF(200 mL) was cooled to −78° C. Ti(OEt)₄ (570 mL, 2.72 mol, 2.0 eq.) wasadded slowly. After completion of the addition, the mixture was stirredfor 1 h, before NaBH₄ (51.3 g, 1.36 mol, 1.0 eq.) was added in portionsover 90 min. The reaction mixture was stirred at −78° C. for 3 h. TLCanalysis showed starting material still remained. EtOH (50 mL) was addedslowly, and the reaction was stirred for 1.5 h and then poured ontobrine (2 L, with 250 mL HOAc) and warmed to r.t. After filtration overCelite, the organic phase was separated and washed with water and brine,dried over anhydrous Na₂SO₄, filtered, and concentrated. The residue waspurified by column chromatography (EtOAc/PE 1:1) to deliver product 12(364 g, 71% yield) as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 8.10 (s,1H), 5.51 (d, J=5.8 Hz, 1H), 5.23-5.15 (m, 1H), 4.41 (q, J=7.0 Hz, 2H),3.48-3.40 (m, 1H), 3.37 (d, J=8.3 Hz, 1H), 2.29 (t, J=13.0 Hz, 1H),1.95-1.87 (m, 1H), 1.73-1.67 (m, 1H), 1.40 (t, J=7.1 Hz, 3H), 1.29 (s,9H), 0.93 (d, J=7.3 Hz, 3H), 0.90 (d, J=7.2 Hz, 3H). MS ESI m/z calcdfor C₁₆H₂₈NaN₂O₄S₂ [M+Na]⁺399.15, found 399.14.

Example 16. Synthesis of Compound 13

To a solution of compound 12 (600 g, 1.60 mol, 1.0 eq.) in ethanol (590mL) was added 4 N HCl in dioxane (590 mL) slowly at 0° C. The reactionwas allowed to warm to r.t. and stirred for 2.5 h. A white precipitatecrushed out and was collected by filtration and washed with EtOAc. Thefiltrate was concentrated and triturated with EtOAc. Two crops of whitesolid were combined and weighed 446 g (90% yield).

Example 17. Synthesis of Compound 14

Compound 10: Azido-Ile-OH (6, 153 g, 0.97 mol, 2.0 eq.) was dissolved inTHF (1.5 L) and cooled to 0° C., to which NMM (214 mL, 1.94 mol, 4.0eq.) and isobutylchloroformate (95 mL, 0.73 mol, 2.0 eq.) were added insequence. The reaction was stirred at 0° C. for 1.0 h. Compound 13 (150g, 0.49 mmol, 1.0 eq.) was added in portions. After stirring at 0° C.for 30 min, the reaction was warmed to r.t. and stirred for 2 h. Waterwas added at 0° C. to quench the reaction and the resulting mixture wasextracted with EtOAc for three times. The combined organic layers werewashed with 1N HCl, saturated NaHCO₃ and brine, dried over anhydrousNa₂SO₄, filtered and concentrated. The residue was purified by columnchromatography (0-30% EtOAc/PE) to give a white solid (140 g, 70%yield). ¹H NMR (500 MHz, CDCl₃) δ 8.14 (s, 1H), 6.57 (d, J=8.9 Hz, 1H),4.91 (d, J=11.1 Hz, 1H), 4.44 (dd, J=13.2, 6.3 Hz, 2H), 4.08-3.95 (m,2H), 2.21 (dd, J=24.4, 11.5 Hz, 2H), 1.90-1.79 (m, 3H), 1.42 (t, J=6.6Hz, 3H), 1.37-1.27 (m, 2H), 1.11 (d, J=6.4 Hz, 3H), 1.01-0.94 (m, 9H).MS ESI m/z calcd for C₁₈H₃₀N₅O₄S [M+H]⁺ 412.19, found 412.19.

Example 18. Synthesis of Compound 15

Compound 11: To a solution of compound 14 (436 g, 1.05 mol, 1.0 eq.) inCH₂Cl₂ (50 mL) was added imidazole (94 g, 1.37 mmol, 1.3 eq.), followedby chlorotriethylsilane (222 mL, 1.32 mol, 1.25 eq.) at 0° C. Thereaction mixture was allowed to warm to r.t. over 1 hour and stirred foran additional hour. Brine was added to the reaction mixture, the organiclayer was separated and the aqueous layer was extracted with EtOAc. Thecombined organic phases were dried, filtered, concentrated under reducedpressure, and purified by column chromatography with a gradient of15-35% EtOAc in petreolum ether to afford product 15 (557.4 g, 95%yield) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 8.12 (s, 1H), 6.75(d, J=8.0 Hz, 1H), 5.20-5.12 (m, 1H), 4.44 (q, J=7.0 Hz, 2H), 4.06-3.97(m, 1H), 3.87 (d, J=3.8 Hz, 1H), 2.14 (d, J=3.8 Hz, 1H), 2.01-1.91 (m,3H), 1.42 (t, J=7.1 Hz, 3H), 1.34-1.25 (m, 2H), 1.06 (d, J=6.8 Hz, 3H),1.00-0.93 (m, 18H), 0.88 (dd, J=19.1, 6.8 Hz, 6H).MS ESI m/z calcd forC₂₄H₄₄N₅O₄SSi [M+H]⁺ 526.28, found 526.28.

Example 19. Synthesis of Compound 16

To a solution of 15 (408 g, 0.77 mol, 1.0 eq.) and methyl iodide (145mL, 2.32 mol, 3.0 eq.) in THF (4 L) was added sodium hydride (60%dispersion in mineral oil, 62.2 g, 1.55 mol, 2.0 eq.) at 0° C. Theresulting mixture was stirred at 0° C. overnight and then poured ontoice-water cooled saturated ammonium chloride (5 L) with vigorousstirring. The mixture was then extracted with EtOAc (3×500 mL) and theorganic layers were dried, filtered, concentrated and purified by columnchromatography with a gradient of 15-35% EtOAc in petreolum ether toafford product 16 (388 g, 93% yield) as a light yellow oil. ¹H NMR (500MHz, CDCl₃) δ 8.09 (s, 1H), 4.95 (d, J=6.6 Hz, 1H), 4.41 (q, J=7.1 Hz,2H), 3.56 (d, J=9.5 Hz, 1H), 2.98 (s, 3H), 2.27-2.06 (m, 4H), 1.83-1.70(m, 2H), 1.41 (t, J=7.2 Hz, 3H), 1.29 (ddd, J=8.9, 6.8, 1.6 Hz, 3H),1.01 (d, J=6.6 Hz, 3H), 0.96 (dt, J=8.0, 2.9 Hz, 15H), 0.92 (d, J=6.6Hz, 3H), 0.90 (d, J=6.7 Hz, 3H). MS ESI m/z calcd for C₂₅H₄₆N₅O₄SSi[M+H]⁺ 540.30, found 540.30.

Example 20. Synthesis of Compound 17

To a solution of compound 16 (1.01 g, 1.87 mmol) in methanol (15 mL) wasadded 0.1N HCl dropwise until a neutral pH was reached. After additionof Pd/C (10 wt %, 583 mg), the mixture was stirred under H₂ (1 atm) atroom temperature for 16 h. The Pd/C was then removed by filtration, withwashing of the filter pad with methanol. The filtrate was concentratedunder reduced pressure and the residue was re-dissolved in EtOAc (50mL), dried over anhydrous Na₂SO₄, filtered and concentrated to affordcompound 17 (900 mg, 94% yield) as a pale yellow oil.

Example 21. Synthesis of Compound 22

To a solution of D-pipecolinic acid (10.0 g, 77.4 mmol, 1.0 eq.) inmethanol (100 mL) was added formaldehyde (37% aqueous solution, 30.8 mL,154.8 mmol, 2.0 eq.), followed by Pd/C (10 wt %, 1.0 g). The reactionmixture was stirred under H₂ (1 atm) overnight, and then filteredthrough Celite, with washing of the filter pad with methanol. Thefiltrate was concentrated under reduced pressure to afford compound 22(10.0 g, 90% yield) as a white solid.

Example 22. Synthesis of Compound 23

To a solution of D-N-methyl pipecolinic acid (2.65 g, 18.5 mmol) inEtOAc (50 mL) were added pentafluorophenol (3.75 g, 20.4 mmol) and DCC(4.21 g, 20.4 mmol). The reaction mixture was stirred at r.t. for 16 h,and then filtered over Celite. The filter pad was washed with 10 mL ofEtOAc. The filtrate was used immediately without further purification orconcentration.

Example 23. Synthesis of Compound 28

A mixture of 2-amino-2-methylpropanoic acid (500 g, 4.85 mol, 1.0 eq.),aqueous formaldehyde (37%, 1.0 L, 12.1 mol, 2.5 eq.) and formic acid(1.0 L) was heated to reflux (80° C.) for 3.0 h. 6 N HCl (850 mL) wasthen added at r.t. and the reaction mixture was concentrated. Theresulting solid was collected by filtration with washing of ethylacetate for three times (1.0 L). The solid was dissolved in water (1.5L) and neutralized to pH 7.0 with 4N NaOH (about 1.0 L solution). Thesolution was concentrated and co-evaporated with ethanol (2.0 L) toremove residual water. MeOH (2.0 L) was added to the residue and thesolid (NaCl) was filtered off with washing of ethyl acetate. Thefiltrate was concentrated under reduced pressure to give a white solid639.2 g, which contains some NaCl and was used without furthertreatment.

Example 24. Synthesis of Compound 29

To a solution of 2-(dimethylamino)-2-methylpropanoic acid (97 g, 0.74mol) in EtOAc (1 L) were added pentafluorophenol (163 g, 0.88 mol) andDIC (126 mL, 0.81 mol). The reaction mixture was stirred at r.t. for 24h, and then filtered over Celite. The filter pad was washed with 10 mLof EtOAc. The filtrate was used immediately without further purificationor concentration.

Example 25. Synthesis of Compound 30

Dry Pd/C (10 wt %, 300 mg) and azide compound 16 (3.33 g, 6.61 mmol)were added to pentafluorophenyl ester 23 in EtOAc. The reaction mixturewas stirred under hydrogen atmosphere for 27 h, and then filteredthrough a plug of Celite, with washing of the filter pad with EtOAc. Thecombined organic portions were concentrated and purified by columnchromatography with a gradient of 0-5% methanol in EtOAc to delivercompound 30 (3.90 g, 86% yield). MS ESI m/z calcd for C₃₂H₅₉N₄O₅SSi[M+H]⁺ 639.39, found 639.39.

Example 26. Synthesis of Compound 31

The coupled product compound 30 (3.90 g, 6.1 mmol) was dissolved inAcOH/water/THF (v/v/v 3:1:1, 100 mL), and stirred at r.t. for 48 h. Thereaction was then concentrated and purified by column chromatography(2:98 to 15:85 MeOH/EtOAc) to afford compound 31 (2.50 g, 72% yield over2 steps). MS ESI m/z calcd for C₂₆H₄₅N₄O₅S [M+H]⁺ 525.30, found 525.33.

Example 27. Synthesis of Compound 32

An aqueous solution of LiOH (0.4N, 47.7 mL, 19.1 mmol, 4.0 eq.) wasadded to a solution of compound 31 (2.50 g, 4.76 mmol, 1.0 eq.) indioxane (47.7 mL) at 0° C. The reaction mixture was stirred at r.t. for2 h and then concentrated. Column chromatography (100% CH₂Cl₂ toCH₂Cl₂/MeOH/NH₄OH 80:20:1) afforded compound 32 (2.36 g, 99% yield) asan amorphous solid. MS ESI m/z calcd for C₂₄H₄₁N₄O₅S [M+H]⁺ 497.27,found 497.28.

Example 28. Synthesis of Compound 33

To a solution of compound 32 (2.36 g, 4.75 mmol) in pyridine (50 mL) at0° C., acetic anhydride (2.25 mL, 24 mmol) was added slowly. Thereaction mixture was allowed to warm to r.t. over 2 h and stirred atr.t. for 24 h. The reaction was concentrated and then treated withdioxane/water (v/v 1:1, 10 mL) for 1 h to destroy possible anhydride.After concentration the residue was purified by column chromatography(100% CH₂Cl₂ to CH₂Cl₂/MeOH/NH₄OH 50:50:1) to afford compound 33 (2.25g, 88% yield) as an amorphous white solid. MS ESI m/z calcd forC₂₆H₄₃N₄O₆S [M+H]⁺ 539.28, found 539.28.

Example 29. Synthesis of Compound 38

To the EtOAc solution of pentafluorophenyl ester 29, compound 16 (200 g,0.37 mol) and dry Pd/C (10 wt %, 10 g) were added. The reaction mixturewas stirred under hydrogen atmosphere (1 atm) for 27 h, and thenfiltered through a plug of Celite, with washing of the filter pad withEtOAc. The combined organic portions were concentrated and purified bycolumn chromatography with a gradient of 0-5% methanol in EtOAc todeliver compound 38 (184 g, 79% yield). MS ESI m/z calcd forC₃₁H₅₈N₄O₅SSi [M+H]⁺ 627.39, found 627.39.

Example 30. Synthesis of Compound 39

Compound 38 (200 g, 0.32 mmol) was dissolved in AcOH/water/THF (v/v/v3:1:1, 638 mL), and stirred at r.t. for 4 days. After the reaction wasconcentrated, toluene was added and concentrated again; this step wasrepeated two times to afford compound 39, which was used directly in thenext step. MS ESI m/z calcd for C₂₅H₄₅N₄O₅S [M+H]⁺ 513.30, found 513.30.

Example 31. Synthesis of Compound 40

An aqueous solution of LiOH (0.4N, 600 mL, 2.55 mol, 8.0 eq.) was addedto a solution of compound 39 (160 g, 0.319 mol, 1.0 eq.) in MeOH (1.2 L)at 0° C. The reaction mixture was stirred at r.t. for 2 h and thenconcentrated. Column chromatography (pure CH₂Cl₂ to 80:20:1CH₂Cl₂/MeOH/NH₄OH) afforded compound 40 (140 g, 910% yield for twosteps) as an amorphous solid. MS ESI m/z calcd for C₂₃H₄₀N₄O₅S [M+H]⁺485.27, found 485.27.

Example 32. Synthesis of Compound 41

A solution of compound 27 (143 g, 0.30 mol, 1.0 eq.) and DMAP (0.36 g,2.95 mmol, 0.01 eq.) in anhydrous THF (1.4 L) and anhydrous DMF (75 mL)was cooled to 0° C., to which TEA (82.2 mL, 0.59 mmol, 2.0 eq.) andacetic anhydride (56 mL, 0.59 mmol, 2.0 eq.) were added. The reactionmixture was allowed to warm to r.t. and stirred for 24 h, and thenconcentrated. Column chromatography (5-50% MeOH/DCM) delivered compound41 (147 g, 95% yield) as an amorphous solid. MS ESI m/z calcd forC₂₅H₄₄N₄O₆S [M+H]⁺ 527.28, found 527.28.

Example 33. Synthesis of Compound 41a

To a solution of compound 41 (5.0 g, 9.5 mmol, 1.0 eq) in anhydrous DCM(100 mL) was added EDC (4.6 g, 23.8 mmol, 2.5 eq) and pentafluorophenol(4.4 g, 23.8 mmol, 2.5 eq) at room temperature under N₂. The mixture wasstirred at room temperature for 2 h, and then diluted in DCM (100 mL),washed with water (2×200 mL) and brine (200 mL), dried over anhydroussodium sulfate, filtered, concentrated and purified by SiO₂ columnchromatography (50% EtOAc/PE) to give compound 41aas a white solid (5.2g, 79% yield) MS ESI m/z calcd for C₃₁H₄₂F₅N₄O₆S [M+H]⁺: 693.27,found:693.27.

Example 34. Synthesis of Compound 95

In a 500 mL round-bottomed flask equipped with a magnetic stir bar wasadded triphenylphosphine (100 g, 381 mmol, 1.0 eq.) and ethyl2-bromopropionate (100 mL, 762 mmol, 2.0 eq.). The mixture was thenheated to 50° C. under N₂ atmosphere overnight. After the white solid(PPh₃) was dissolved, a large amount of white solid was generated.Trituration with petroleum ether/EtOAc and filtration gave compound 95as a white solid (135 g, 80% yield). MS ESI m/z calcd for C₂₃H₂₄O₂P[M-Br]⁺363.15, found 363.13.

Example 35. Synthesis of Compound 96

A solution of compound 95 (135.42 g, 305.7 mmol) in dichloromethane (500mL) was added slowly into 10% NaOH solution (450 mL) with vigorousstirring. The organic solution rapidly turned bright yellow. After 30minutes, TLC analysis showed that the reaction was completed. Layerswere separated and the aqueous layer was further extracted with CH₂Cl₂(2×200 mL). Combined organic layers were washed with brine, dried overanhydrous Na₂SO₄ and concentrated to give a yellow solid 96 (104 g, 94%yield). MS ESI m/z calcd for C₂₃H₂₄O₂P [M+H]⁺ 362.14, found 363.13. Thecrude product was used directly in the next step.

Example 36. Synthesis of Compound 98

To a mixture of Boc-L-Tyr-OMe (670 g, 2.27 mol, 1.0 eq.), K₂CO₃ (358 g,2.5 mol, 1.1 eq.) and KI (38 g, 0.227 mol, 0.1 eq.) in acetone (3 L) wasadded benzyl bromide (283 mL, 2.38 mol, 1.05 eq.) slowly. The mixturewas then refluxed overnight. Water (6 L) was added and the reactionmixture was extracted with EtOAc (5×100 L). The combined organic layerswere washed with brine (2 L), dried over anhydrous Na₂SO₄, filtered,concentrated and purified by SiO₂ column chromatography (4:1hexanes/EtOAc) to give a white solid 98 (795 g, 91% yield). ¹H NMR (500MHz, CDCl₃) δ 7.43 (d, J=7.0 Hz, 2H), 7.38 (t, J=7.4 Hz, 2H), 7.32 (t,J=7.2 Hz, 1H), 7.04 (d, J=8.5 Hz, 2H), 6.91 (d, J=8.6 Hz, 2H), 5.04 (s,2H), 4.55 (d, J=6.9 Hz, 1H), 3.71 (s, 3H), 3.03 (qd, J=14.0, 5.8 Hz,2H), 1.43 (s, 9H). ESI: m/z: calcd for C₂₂H₂₈NO₅ [M+H]⁺: 386.19, found386.19.

Example 37. Synthesis of Compound 99

To a solution of ester 98 (380 g, 987 mmol, 1.0 eq.) in anhydrousdichloromethane (1 L) at −78° C. was added DIBAL (1.0 M in hexanes, 2.9L, 2.9 eq.) over 3 h. After the addition was completed, the mixture wasquenched with 3 L of ethanol. 1N HCl was added dropwise until pH 4 wasreached. The resulting mixture was allowed to warm to 0° C. Layers wereseparated and the aqueous layer was further extracted with EtOAc (3×3L). The combined organic solution was washed with brine, dried overanhydrous Na₂SO₄, and concentrated. Trituration with PE/EtOAc andfiltration gave a white solid 99 (263 g, 75% yield). 1H NMR (500 MHz,CDCl₃) δ 9.65 (s, 1H), 7.45 (d, J=7.1 Hz, 2H), 7.41 (t, J=7.4 Hz, 2H),7.35 (t, J=7.1 Hz, 1H), 7.11 (d, J=8.6 Hz, 2H), 6.95 (d, J=8.6 Hz, 2H),5.07 (s, 2H), 4.42 (dd, J=12.4, 6.1 Hz, 1H), 3.09 (d, J=6.2 Hz, 2H),1.46 (s, 9H). ESI: m/z: calcd for C₂₁H₂₆NO₄ [M+H]⁺: 356.18, found356.19.

Example 38. Synthesis of Compound 100

To a solution of aldehyde 99 (81.4 g, 229 mmol, 1.0 eq.) in anhydrousdichloromethane (800 mL) at room temperature was added ylide 96 (2.0eq.) in anhydrous dichloromethane (800 mL) over 30 min. The mixture wasstirred at room temperature overnight then concentrated and purified bySiO₂ column chromatography (6:1 petroleum ether/EtOAc) to give a whitesolid 100 (63.4 g, 63% yield). ¹H NMR (500 MHz, CDCl₃) δ 7.45-7.41 (m,2H), 7.40-7.35 (m, 2H), 7.33 (d, J=7.2 Hz, 1H), 7.10-7.06 (m, 2H),6.92-6.88 (m, 2H), 6.50 (dd, J=8.8, 1.3 Hz, 1H), 5.04 (s, 2H), 4.57 (s,2H), 4.18 (q, J=7.1 Hz, 2H), 2.86 (d, J=8.5 Hz, 1H), 2.72 (dd, J=13.6,6.8 Hz, 1H), 1.71 (d, J=1.4 Hz, 3H), 1.41 (d, J=2.2 Hz, 9H), 1.28 (td,J=7.5, 5.1 Hz, 4H). MS ESI m/z calcd for C₂₆H₃₃NaNO₅ [M+Na]⁺462.24,found 462.22.

Example 39. Synthesis of Compound 101

In a hydrogenation bottle, Pd/C (1.83 g, 10 wt %, 50% water) was addedto a solution of compound 100 (30.2 g, 68.9 mmol) in THF (100 mL) andmethanol (300 ml). The mixture was shaken under 1 atm H₂ overnight,filtered through Celite (filter aid), and the filtrate was concentratedto afford compound 101 (25.0 g, theoretical yield) as a colorless oil.¹H NMR (500 MHz, CDCl₃) δ 6.99 (d, J=7.0 Hz, 2H), 6.72 (d, J=7.6 Hz,2H), 4.39 (s, 1H), 4.18-4.04 (m, 2H), 3.82 (s, 1H), 2.60 (dd, J=37.2,20.9 Hz, 4H), 1.95-1.81 (m, 1H), 1.39 (s, 11H), 1.24 (dd, J=9.5, 4.3 Hz,3H), 1.13 (t, J=8.9 Hz, 3H). MS ESI m/z calcd for C₁₉H₃₁NO₅ [M+H]⁺352.20, found 352.19.

Example 40. Synthesis of Compound 102

To a solution of compound 101 (5.96 g, 35.9 mmol, 1.0 eq.) in anhydrousdichloromethane (200 mL)was added Ac₂O (3.2 mL, 33.9 mmol, 2.0 eq.) andHNO₃ (65%-68%, 3.5 mL, 50.79 mmol, 3.0 eq.) at room temperature. Themixture was stirred at room temperature for 30 min, and TLC analysisshowed that the reaction was completed. The reaction solution was washedwith water (3×200 mL), and the aqueous layer was back-extracted withdichloromethane (3×100 mL). The combined dichloromethane solution waswashed with brine, dried over anhydrous Na₂SO₄, filtered, concentratedand purified by SiO₂ column chromatography (5:1 hexanes/EtOAc) to givecompound 102 as a yellow solid (4.18 g, 72% yield). ¹H NMR (500 MHz,CDCl₃) δ 10.49 (s, 1H), 7.89 (s, 1H), 7.44 (d, J=8.4 Hz, 1H), 7.09 (d,J=8.6 Hz, 1H), 4.32 (d, J=8.3 Hz, 1H), 4.12 (dd, J=14.0, 7.0 Hz, 2H),3.80 (s, 1H), 2.76 (dd, J=13.0, 6.8 Hz, 2H), 2.59 (s, 1H), 1.88 (s, 1H),1.37 (t, J=8.7 Hz, 9H), 1.25 (dd, J=13.5, 6.9 Hz, 4H), 1.16 (t, J=8.0Hz, 3H). MS ESI m/z calcd for C₁₉H₂₈NaN₂O₇ [M+Na]⁺419.19, found 419.17.

Example 41. Synthesis of Compound 103

To a solution of ester 102 (15.3 g, 38.6 mmol, 1.0 eq.) in THF (100 mL)and methanol (100 mL) was added LiOH—H₂O (16.3 g, 389 mmol, 10.0 eq.) inwater (190 mL) at room temperature. The mixture was stirred at roomtemperature for 40 min. and then diluted with water (400 mL) and 1NKHSO₄ was added dropwise until pH 3-4 was reached. After extraction withEtOAc (3×300 mL), the organic phase was washed with brine, dried overanhydrous Na₂SO₄, filtered, concentrated to give 103 as a yellow solid(14.4 g, theoretical yield). ¹H NMR (500 MHz, CDCl₃) δ 10.48 (s, 1H),7.98-7.88 (m, 1H), 7.42 (dd, J=18.4, 8.2 Hz, 1H), 7.14-7.03 (m, 1H),4.48 (d, J=8.6 Hz, 1H), 3.90 (s, 1H), 2.82-2.53 (m, 3H), 1.97-1.82 (m,2H), 1.42-1.27 (m, 1OH), 1.21 (d, J=6.7 Hz, 4H). MS ESI m/z calcd forC₁₇H₂₃N₂O₇ [M−H]⁻ 367.16, found 367.14.

Example 42. Synthesis of Compound 104

In a hydrogenation bottle, Pd/C (2.60 g, 10 wt %, 50% water) was addedto a solution of compound 103 (26.0 g, 70.6 mmol, 1.0 eq.) in methanol(260 mL). The mixture was shaken overnight under 1 atm H₂ then filteredthrough Celite (filter aid), the filtrate was concentrated to affordcompound 104 as a green oil (24.0 g, >100% yield).

Example 43. Synthesis of Compound 106

A mixture of tert-butyl-2-bromopropanoate (255 g, 1.22 mol, 1.0 eq.) andtriphenyl phosphine (320 g, 1.22 mol, 1.0 eq.) in dry acetonitrile (1 L)was stirred at room temperature for 18 h. Acetonitrile was removed underreduced pressure and toluene was added to crash out a white precipitate.Toluene was then decanted off and the white solid was dissolved indichloromethane (1 L) and transferred to a separatory funnel. 10% NaOH(1 L) was added to the funnel, and the organic layer immediately turnedyellow after shaking. The organic layer was separated and the aqueouslayer was extracted with dichloromethane (1 L) once. The dichloromethanelayers were combined and washed with brine (400 mL) once, then driedover Na₂SO₄, filtered and concentrated, giving the ylide 106 as a yellowsolid (280 g, 58%).

Example 44. Synthesis of Compound 107

Aldehyde 99 (450 g, 1.27 mol, 1.0 eq.) was dissolved in drydichloromethane (3 L), to which tert-butyl ester ylide 106 (546 g, 1.40mmol, 1.1 eq.) was added and the solution was stirred at r.t. overnightas determined complete by TLC. Purification by column chromatography(10-50% EtOAc/hexanes) afforded compound 107 (444 g, 75% yield) as awhite solid. ESI m/z calcd for C₂₈H₃₈NO₅ [M+H]⁺: 468.27, found 468.22.

Example 45. Synthesis of Compound 108

Compound 107 (63 g, 0.13 mol) was dissolved in methanol (315 mL) andhydrogenated (1 atm H₂) with Pd/C catalyst (10 wt %, 6.3 g) at r.t.overnight. The catalyst was filtered off and the filtrate wereconcentrated under reduced pressure to afford compound 108 (45.8 g, 93%yield).

Example 46. Synthesis of Compound 109

To a solution of compound 108 (390 g, 1.03 mol, 1.0 eq.) in THF (4 L)tert-butyl nitrite (1.06 kg, 10.3 mol, 10 eq.) was added at r.t. and thereaction was stirred overnight. After removal of THF, the residue waspurified by column chromatography (10-50% EtOAc/hexanes) to affordcompound 109 (314 g, 72% yield) as a light yellow solid.

Example 47. Synthesis of Compound 110

To a solution of 109 (166 g, 0.392 mol, 1.0 eq.) in EtOAc (500 mL) wasadded Pd/C (10 wt %, 16 g) under nitrogen, and the reaction flask wasevacuated and purged with hydrogen for 3 times. The reaction mixture wasstirred under hydrogen (1 atm) at r.t. for 16 h and then filtered overCelite and concentrated to afford product 110 (146 g, 97% yield) as alight yellow foam. ¹H NMR (400 MHz, CDCl₃) δ 6.62 (d, J=7.9 Hz, 1H),6.55 (s, 1H), 6.43 (d, J=7.3 Hz, 1H), 4.39 (dd, J=53.0, 44.2 Hz, 1H),3.77 (s, 4H), 2.72-2.29 (m, 3H), 1.83-1.58 (m, 1H), 1.40 (d, J=7.6 Hz,18H), 1.24 (s, 1H), 1.06 (t, J=5.7 Hz, 3H). MS ESI m/z calcd forC₂₁H₃₅N₂O₅ [M+H]⁺ 394.25, found 395.25.

Example 48. Synthesis of Compound 114

To a solution of (S)-4-isopropyloxazolidin-2-one (5.00 g, 38.7 mmol, 1.0eq.) in anhydrous THF (200 mL)at −78° C. was added n-BuLi (2.5 M inhexanes, 17.0 mL, 1.2 eq.) in 30 min under N₂. The mixture was stirredat −78° C. for 1 h, and then propionyl chloride (4.0 mL, 42.58 mmol, 1.1eq.) was added dropwise. After the mixture was stirred at −78° C. foranother 1 h, TLC analysis indicated the reaction completed. Saturatedammonium chloride solution (250 mL) was added and extracted with EtOAc(3×100 mL). The combined organic layers were washed with 1N NaOHsolution (200 mL) and brine (300 mL), dried over anhydrous Na₂SO₄,filtered, concentrated and purified by column chromatography (7:1hexanes/EtOAc) to give compound 114 as a colorless oil (6.36 g, 89%yield).MS ESI m/z calcd for C₉H₁₆NO₃ [M+H]⁺ 186.10, found 186.10. ¹H NMR(400 MHz, CDCl₃) δ 4.48-4.39 (m, 1H), 4.27 (t, J=8.7 Hz, 1H), 4.21 (dd,J=9.1, 3.1 Hz, 1H), 3.06-2.82 (m, 2H), 2.38 (dtd, J=14.0, 7.0, 4.0 Hz,1H), 1.17 (t, J=7.4 Hz, 3H), 0.90 (dd, J=17.0, 7.0 Hz, 6H).

Example 49. Synthesis of Compound 115

To a solution of (S)-4-isopropyl-3-propionyloxazolidin-2-one (2.00 g,11.9 mmol, 1.1 eq.) in anhydrous dichloromethane (20 mL)at 0° C. wasadded DIPEA (2.3 mL, 12.9 mmol, 1.2 eq.) and n-Bu₂BOTf (1.0 M indichloromethane, 12.0 mL, 1.1 eq.) under N₂. The mixture was stirred at0° C. for 45 min, then cooled to −78° C., to which a solution ofcompound 99 (4.24 mL, 10.8 mmol, 1.0 eq.) in dichloromethane was addeddropwise. The mixture was stirred at −78° C. for 1 h and then warmedslowly to room temperature. The mixture was stirred at room temperatureovernight, and PBS (0.1M, pH 7.0, 100 mL) was added. After phaseseparation, the aqueous phase was further extracted with dichloromethane(3×50 mL). The combined organic layers were washed with brine (200 mL),dried over anhydrous Na₂SO₄, filtered and concentrated. The crudeproduct was re-dissolved in methanol (100 mL)and treated with H₂O₂ (30%aqueous solution, 26 mL, 23 eq.) at 0° C. for 3 h. The methanol wasremoved by rotary evaporation and water (100 mL) was added. Theresulting mixture was extracted with EtOAc (3×100 mL). The combinedorganic layers were washed with brine (300 mL), dried over anhydrousNa₂SO₄, filtered, concentrated and purified by SiO₂ columnchromatography (3:1 hexanes/EtOAc) to give compound 115 as a foamysolid(2.70 g, 49% yield). ¹H NMR (400 MHz, CDCl₃) δ 6.52-7.26 (m, 5H),7.15 (d, J=7.4 Hz, 2H), 6.93 (d, J=7.3 Hz, 2H), 5.05 (s, 2H), 4.69 (d,J=7.0 Hz, 1H), 4.47 (s, 1H), 4.36 (t, J=7.8 Hz, 1H), 4.17 (d, J=8.5 Hz,1H), 3.93 (d, J=7.1 Hz, 1H), 3.85 (s, 2H), 2.84 (d, J=6.9 Hz, 2H), 2.31(s, 1H), 1.40-1.37 (m, 9H), 1.31 (s, 3H), 0.92 (dd, J=13.4, 6.6 Hz, 6H).MS ESI m/z calcd for C₃₀H₄₁N₂O₇ [M+H]⁺ 541.28, found 541.30.

Example 50. Synthesis of Compound 116

A mixture of compound 115 (2.50 g, 4.63 mmol, 1.0 eq.) and1,1′-thiocarbonyldiimidazole (2.48 g, 13.89 mmol, 3.0 eq.) in anhydrousTHF (46 mL) was refluxed overnight. Water (100 mL) was added and theresulting mixture was extracted with EtOAc (3×50 mL). The combinedorganic layers were washed with brine (200 mL), dried over anhydrousNa₂SO₄, filtered, concentrated and purified by SiO₂ columnchromatography (3:1 hexanes/EtOAc) to give compound 116 as a yellow foam(2.33 g, 77% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.41 (s, 1H), 7.67 (s,1H), 7.36 (dt, J=16.0, 6.9 Hz, 6H), 7.09 (s, 1H), 7.05 (d, J=8.4 Hz,2H), 6.86 (d, J=8.4 Hz, 2H), 6.32 (d, J=9.5 Hz, 1H), 5.01 (s, 2H),4.56-4.43 (m, 2H), 4.32 (ddd, J=16.2, 15.6, 7.8 Hz, 3H), 4.19 (d, J=8.7Hz, 1H), 2.96 (dd, J=14.6, 4.4 Hz, 1H), 2.49 (dd, J=14.5, 10.5 Hz, 1H),2.29 (td, J=13.4, 6.7 Hz, 1H), 1.31 (s, 3H), 1.29 (s, 9H), 0.91 (dd,J=13.9, 6.9 Hz, 6H). MS ESI m/z calcd for C₃₄H₄₃N₄O₇S[M+H]⁺ 651.27,found 651.39.

Example 51. Synthesis of Compound 117

To a solution of compound 116 (1.90 g, 2.92 mmol, 1.0 eq.) in anhydroustoluene (30 mL)was added n-Bu₃SnH (1.6 mL, 5.84 mmol, 2.0 eq.) andazodiisobutyronitrile (0.05 g, 0.584 mmol, 0.1 eq.) in sequence. Themixture was refluxed for 2.5 h and then concentrated and purified bySiO₂ column chromatography (5:1 hexanes/EtOAc) to give compound 117 as awhite foam (1.21 g, 79% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.36 (ddd,J=24.5, 14.5, 7.1 Hz, 5H), 7.08 (d, J=8.5 Hz, 2H), 6.90 (d, J=8.5 Hz,2H), 5.04 (d, J=5.1 Hz, 2H), 4.48 (d, J=4.2 Hz, 1H), 4.33 (t, J=8.4 Hz,1H), 4.22 (d, J=9.7 Hz, 1H), 4.15 (d, J=8.8 Hz, 1H), 3.81 (s, 2H), 2.73(dd, J=14.1, 5.9 Hz, 1H), 2.61 (dd, J=14.0, 7.2 Hz, 1H), 2.29 (dq,J=13.5, 6.8 Hz, 1H), 2.11-2.00 (m, 1H), 1.35 (s, 9H), 1.20 (d, J=6.9 Hz,3H), 0.89 (dd, J=14.0, 6.9 Hz, 6H). MS ESI m/z calcd for C₃₀H₄₁N₂O₆[M+H]⁺ 525.28, found 525.37.

Example 52. Synthesis of Compound 118

To a solution of compound 117 (1.20 g, 2.29 mmol, 1.0 eq) in THF (30 mL)were added LiOH (0.192 g, 4.58 mmol, 2.0 eq.) in water (6 mL) and H₂O₂(30% aqueous solution, 1.4 mL, 6.0 eq.). After 3 h of stirring at 0° C.,sodium bisulfite solution (1.5 M, 30 mL) was added to quench thereaction. After 30 min, 1 N KHSO₄ was added dropwise until pH 4 wasreached. The reaction mixture was then extracted with EtOAc (3×50 mL).The EtOAc solution was washed with brine, dried over anhydrous Na₂SO₄,filtered, concentrated and purified by SiO₂ column chromatography (3:1hexanes/EtOAc, containing 1% HOAc) to give compound 118 as a white solid(0.78 g, 82% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.46-7.28 (m, 5H), 7.07(d, J=7.7 Hz, 2H), 6.91 (d, J=7.8 Hz, 2H), 4.52 (d, J=8.5 Hz, 1H), 3.87(d, J=41.8 Hz, 1H), 2.82-2.43 (m, 3H), 1.85 (t, J=12.2 Hz, 1H), 1.41 (s,9H), 1.17 (d, J=6.9 Hz, 3H). MS ESI m/z calcd for C₂₄H₃₂NO₅ [M+H]⁺414.22, found 414.21.

Example 53. Synthesis of Compound 119

A mixture of compound 118 (0.77 g, 1.86 mmol, 1.0 eq.) and Pd/C (10%,0.25 g) in methanol (15 mL) was hydrogenated under 1 atm H₂ pressure for16 h and then filtered through Celite (filter aid). The filtrate wasconcentrated to afford compound 119 as a white solid (0.58 g, 96%yield). ¹H NMR (400 MHz, CDCl₃) δ 7.00 (d, J=7.5 Hz, 2H), 6.80 (s, 2H),4.51 (d, J=9.0 Hz, 1H), 3.88 (s, 1H), 2.66 (dd, J=65.6, 22.6 Hz, 4H),1.88 (t, J=12.2 Hz, 1H), 1.42 (s, 9H), 1.14 (d, J=6.6 Hz, 3H). MS ESIm/z calcd for C₁₇H₂₆NO₅ [M+H]⁺: 324.17, found 324.16.

Example 54. Synthesis of Compound 120

To a solution of compound 119 (0.57 g, 1.76 mmol, 1.0 eq.) in THF (10mL)was added t-BuONO (0.63 mL, 5.28 mmol, 3.0 eq.) at 0° C. The reactionwas stirred at 0° C. for 1 hr then room temperature 1 h. After water (50mL) was added, the reaction mixture was extracted with EtOAc (3×30 mL).The combined organic layers were washed with brine (100 mL), dried overanhydrous Na₂SO₄, filtered, concentrated and purified by SiO₂ columnchromatography (2:1 hexanes/EtOAc, containing 1% HOAc) to give compound120 as a yellow solid (0.50 g, 77% yield). ¹H NMR (400 MHz, DMSO) δ 7.92(s, 1H), 7.47 (d, J=8.3 Hz, 1H), 7.05 (d, J=8.5 Hz, 1H), 3.73 (s, 1H),2.78 (dd, J=13.6, 5.3 Hz, 1H), 2.69-2.47 (m, 2H), 1.87 (t, J=11.9 Hz,1H), 1.47-1.37 (m, 1H), 1.32 (s, 9H), 1.17 (d, J=7.2 Hz, 3H). MS ESI m/zcalcd for C₁₇H₂₅N₂O₇ [M+H]⁺ 369.15, found 369.14.

Example 55. Synthesis of Compound 121

A mixture of compound 120 (0.50 g, 1.36 mmol, 1.0 eq.) and Pd/C (10 wt%, 0.02 g) in methanol (10 mL) was hydrogenated (1 atm H₂) at r.t. for 1h, and then filtered through Celite (filter aid). The filtrate wasconcentrated to afford compound 121 as a white foam (0.43 g, 93% yield).MS ESI m/z calcd for C₁₇H₂₇N₂O₅ [M+H]⁺ 339.18, found 339.17. ¹H NMR (400MHz, MeOD) δ 6.60 (d, J=7.9 Hz, 2H), 6.44 (d, J=7.3 Hz, 1H), 3.71 (d,J=6.3 Hz, 1H), 2.62-2.37 (m, 3H), 1.83 (ddd, J=13.7, 9.9, 3.7 Hz, 1H),1.39 (s, 9H), 1.13 (d, J=7.1 Hz, 3H).

Example 56. Synthesis of Compound 124

To a solution of maleic anhydride (268 g, 2.73 mol) in acetic acid (1 L)was added 4-aminobutanoic acid (285 g, 2.76 mol). After stirring at r.t.for 30 min, the reaction was refluxed for 1.5 h, cooled to r.t. andevaporated under vacuum to give a residue, which was taken up in EA,washed with water and brine, and dried over anhydrous Na₂SO₄, filteredand concentrated. The crude product was crystallized from EtOAc and PEto give a white solid (400 g, 80% yield). 1H NMR (500 MHz, CDCl₃) δ 6.71(s, 2H), 3.60 (t, J=6.7 Hz, 2H), 2.38 (t, J=7.3 Hz, 2H), 2.00-1.84 (m,2H).

Example 57. Synthesis of Compound 125

Compound 124 (400 g, 2.18 mol, 1.0 eq.) was dissolved in CH₂Cl₂ (1.5 L),to which N-hydroxysuccinimide (276 g, 2.40 mmol, 1.1 eq.) and DIC (303g, 2.40 mol, 1.1 eq.) were added at r.t. and stirred overnight. Thereaction was concentrated and purified by column chromatography (1:2petroleum ether/EtOAc) to give NHS ester 125 as a white solid (382 g,63% yield). ¹H NMR (500 MHz, CDCl₃) δ 6.74 (s, 2H), 3.67 (t, J=6.8 Hz,2H), 2.85 (s, 4H), 2.68 (t, J=7.5 Hz, 2H), 2.13-2.03 (m, 2H).

Example 58. Synthesis of Compound 126

To a solution of 124 (60 g, 328 mmol, 1.3 eq.) in THF (600 mL) was addedNMM (85.3 mL, 984 mmol, 3.0 eq.) at 0° C. with stirring, followed byisobutyl chloroformate (44.6 mL, 426 mmol, 1.3 eq.) dropwise. Afterstirring at 0° C. for 2 h, the resulting mixture was added dropwise to asolution of 104 (102 g, 259 mmol, 1.0 eq.) in THF (400 mL) while keepingthe temperature at 0° C. After the addition was completed, the reactionwas stirred for additional 30 min. and then quenched with water (300mL), extracted with EtOAc (3×300 mL). The combined organic layers weredried, filtered, concentrated and purified by column chromatography witha gradient of 9-35% EtOAc/PE to afford compound 126 (104 g, 73% yield)as a light yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.86 (s, 1H), 8.40(d, J=17.3 Hz, 1H), 6.87 (s, 3H), 6.70 (s, 2H), 4.53-4.16 (m, OH), 3.79(s, 1H), 3.62 (t, J=6.1 Hz, 1H), 2.63 (s, 1H), 2.40 (t, J=6.9 Hz, 1H),2.12-1.88 (m, 4H), 1.84-1.64 (m, 1H), 1.38 (t, J=9.6 Hz, 6H), 1.06 (t,J=6.0 Hz, 3H).

Example 59. Synthesis of Compound 127

Compound 126 (12.7 g, 22.7 mmol) dissolved in CH₂Cl₂ (20 mL) was treatedwith TFA (40 mL) at 0° C. and the reaction was warmed to r.t. andstirred for 3h. The mixture was concentrated and co-evaporated withtoluene three times. The residue was triturated with diethyl ether and alight yellow solid 127 was collected (11.4 g, theoretical yield).

Example 60. Synthesis of Compound 128

To a solution of carboxylic acid 33 (40 mg, 0.074 mmol, 1.0 eq.) inEtOAc was added pentafluorophenol (27 mg, 0.148 mmol, 2.0 eq.) and DCC(23 mg, 0.111 mmol, 1.5 eq.). The reaction mixture was stirred at r.t.for 16 h and then filtered over a Celite pad, with washing of the padwith EtOAc. The filtrate was concentrated and re-dissolved in DMA (6mL), then compound 127 (56.6 mg, 0.13 mmol) and DIPEA (47.4 μL, 0.18mmol) were added. The reaction mixture was stirred at r.t. for 24 h andthen concentrated and purified by reverse phase HPLC (C₁₈ column,10-100% acetonitrile/water) to afford compound 128 (43 mg, 63% yield) asa white solid. MS ESI m/z calcd for C₄₆H₆₆N₇O₁₁S [M+H]⁺ 924.45, found924.45.

Example 61. Synthesis of Compound 132

To a solution of compound 41a (11 g, 15.9 mmol, 1.0 eq.) and compound127 (12.3 g, 23.8 mmol, 1.5 eq.) in DMF (100 mL) was added DIPEA (6.9mL, 39.7 mmol, 2.5 eq.) at 0° C. The reaction mixture was warmed to r.t.and stirred for 1 h. The mixture was concentrated under vacuum andpurified on silica gel column (100% DCM to 10% MeOH/DCM) to givecompound 132 (10 g, 69% yield) as an amorphous solid. MS ESI m/z calcdfor C₄₅H₆₅N₇O₁₁S [M+H]⁺ 912.45, found 912.45.

Example 62. Synthesis of Compound 166

To a solution of (R)—4-isopropyloxazolidin-2-one (25.0 g, 0.194 mol, 1.0eq) in anhydrous THF (1150 mL) was added n-BuLi (85.0 mL, 0.213 mol, 1.1eq) at −78° C. under N₂ and the mixture was stirred at the sametemperature for 1 h, a large number of white solids formed. Thenpropionyl chloride (20.0 mL, 0.232 mol, 1.2 eq) was added at −78° C. andthe mixture was stirred at the same temperature for 1 h. After theconsumption of (S)-4-isopropyloxazolidin-2-one monitored by TLC, thesolution was poured into saturated ammonium chloride solution (1.2 L)and the mixture was extracted with EA (700 mL, 350 mL×2). The organicextract was washed with 1.0N NaOH solution (1.0 L) and brine (1.0 L),dried over anhydrous sodium sulfate, filtered, concentrated in vacuo andpurified by SiO₂ column chromatography (PE:EA=10:1) to give the titlecompound as a colorless oil (32.6 g, 90.8%). ESI m/z: calcd for C₉H₁₇NO₃[M+H]⁺: 186.1, found 186.1. ¹H NMR (400 MHz, CDCl₃) δ 4.48-4.37 (m, 1H),4.27 (t, J=8.7 Hz, 1H), 4.21 (dd, J=9.1, 3.1 Hz, 1H), 3.04-2.82 (m, 2H),2.45-2.30 (m, 1H), 1.17 (t, J=7.4 Hz, 3H), 0.90 (dd, J=17.1, 7.0 Hz,6H).

Example 63. Synthesis of Compound 167

To a solution of (R)—4-isopropyl-3-propionyloxazolidin-2-one (18.4 g,99.5 mmol, 1.1 eq) in anhydrous DCM (200 mL) were added Bu₂BOTf (1 Mdichloromethane solution, 100 mL, 100 mmol, 1.1 eq) and DIPEA(19 mL,108.6 mmol, 1.2 eq) at 0° C. under N₂, and the mixture was stirred atthe same temperature for 45 min. A solution of aldehyde 99(32.2 g, 90.5mmol, 1.0 eq) in dichloromethane (320 mL) was added at −78° C. andstirred at the same temperature for 1 h, then the solution was allowedto slowly warm to room temperature for 15 hours. The mixture was pouredinto 700 mL of potassium phosphate buffer (pH 7.0) and extracted withethyl acetate. The organic extract was washed with brine, dried overanhydrous sodium sulfate, filtered, and then concentrated in vacuo. Theresidue was dissolved in methanol (730 mL) and cooled to 0° C., then 30%H₂O₂ aqueous solution (225 mL) was added slowly, and the mixture wasstirred at the same temperature for 3 hours. After addition of water(750 mL), the mixture was concentrated in vacuo to remove methanol. Theresulting aqueous solution was extracted with ethyl acetate (500 mL, 150mL×2), and the organic extract was washed with 5% sodium hydrogencarbonate solution and brine, dried over anhydrous sodium sulfate,filtered, concentrated in vacuo and purified by SiO₂ columnchromatography (PE:EA=3:1) to give the title compound as a white foam(31.7 g, 64.8%).ESI m/z: calcd for C₃₀H₄₁N₂O₇ [M+H]⁺: 541.3, found 541.3o ¹H NMR (400 MHz, CDCl₃) δ 7.49-7.29 (m, 5H), 7.17 (t, J=10.7 Hz, 2H),6.93 (d, J=7.0 Hz, 2H), 5.06 (s, 2H), 4.28 (dd, J=44.4, 36.4 Hz, 3H),4.04-3.52 (m, 3H), 3.11-2.73 (m, 2H), 2.35 (s, 1H), 1.41 (t, J=16.3 Hz,9H), 0.91 (dd, J=15.6, 6.4 Hz, 5H).

Example 64. Synthesis of Compound 168

To a solution of compound 167 (28.3 g, 52.3 mmol, 1.0 eq) in anhydrousTHF (350 mL) was added 1,1-thiocarbonyl diimidazole (TCDI) (35.1 g,157.0 mmol, 3.0 eq), and the mixture was heated under reflux overnight.After the consumption of starting material monitored by TLC, the mixturewas concentrated in vacuo and purified by SiO₂ column chromatography(PE:EA=3:1) to give the title compound as a pale yellow foam (26.1 g,76.8%). ESI m/z: calcd for C₃₄H₄₃N₄O₇S [M+H]⁺: 651.3, found 651.3 o ¹HNMR (400 MHz, CDCl₃) δ 8.21 (s, 1H), 7.43 (d, J=11.8 Hz, 1H), 7.42-7.28(m, 5H), 7.06 (d, J=8.3 Hz, 2H), 7.01 (s, 1H), 6.80 (d, J=8.3 Hz, 2H),6.17 (dd, J=8.5, 2.9 Hz, 1H), 4.96 (s, 2H), 4.42-4.04 (m, 5H), 2.83 (dd,J=14.2, 6.2 Hz, 1H), 2.69 (dd, J=14.2, 7.1 Hz, 1H), 2.32 (dd, J=6.8, 4.2Hz, 1H), 1.37 (s, 9H), 1.30 (d, J=6.9 Hz, 3H), 0.87 (dd, J=9.9, 7.0 Hz,6H).

Example 65. Synthesis of Compound 169

To a solution of compound 168(26.0 g, 40.0 mmol, 1.0 eq) in anhydroustoluene (350 mL) was added n-Bu₃SnH (21.5 mL, 80.0 mmol, 2.0 eq) and2,2′-azobis(2-methylpropionitrile) (AIBN) (0.066 g, 0.01 eq) under N₂,and the mixture was heated under reflux for 1 hour. After theconsumption of starting material monitored by TLC, the mixture wasconcentrated in vacuo and purified by SiO₂ column chromatography(PE:EA=5:1) to give the title compound as a white foam (6.0 g, 37.3%).ESI m/z: calcd for C₃₀H₄₁N₂O₆ [M+H]⁺: 525.3, found 525.3 o ¹H NMR (400MHz, CDCl₃) δ 7.37 (ddd, J=25.1, 15.1, 7.1 Hz, 5H), 7.08 (d, J=7.9 Hz,2H), 6.89 (d, J=8.4 Hz, 2H), 5.03 (s, 2H), 4.61 (d, J=8.4 Hz, 1H), 4.40(s, 1H), 4.32-4.08 (m, 2H), 3.91-3.66 (m, 2H), 2.83 (d, J=8.4 Hz, 1H),2.60 (t, J=10.1 Hz, 1H), 2.33 (s, 1H), 1.71 (s, 1H), 1.41 (s, 9H), 1.15(d, J=6.5 Hz, 3H), 0.87 (dd, J=17.0, 7.0 Hz, 6H).

Example 66. Synthesis of Compound 170

To a solution of compound 169(7.84 g, 15.0 mmol, 1.0 eq) in THF (90 mL)and water(30 mL) was added LiOH·H₂O (1.57 g, 37.5 mmol, 2.5 eq) in 30%H₂O₂ aqueous solution (11.4 mL, 112.5 mmol, 7.5 eq) at 0° C., and themixture was stirred at the same temperature for 3 hours. After additionof 1.5M Na₂SO₃ solution (160 mL) at 0° C., the mixture was stirred atthe same temperature for 30 min. then 1N KHSO₄ was added slowly until pH4. The resulting aqueous solution was extracted with EA (200 mL, 75mL×2), and the organic extract was washed with brine, dried overanhydrous sodium sulfate, filtered, concentrated in vacuo and purifiedby SiO₂ column chromatography (PE:EA=2:1) to give the title compound asa white solid (6.18 g, 100%). ESI m/z: calcd for C₂₄H₃₂N₁O₅ [M+H]⁺:414.2, found 414.2. ¹H NMR (400 MHz, CDCl₃) δ 7.39 (ddd, J=24.5, 15.0,7.2 Hz, 5H), 7.11 (d, J=7.8 Hz, 2H), 6.93 (d, J=8.3 Hz, 2H), 5.06 (s,2H), 4.44 (t, J=8.3 Hz, 1H), 3.83 (d, J=69.4 Hz, 1H), 2.85-2.61 (m, 2H),2.61-2.40 (m, 1H), 1.99-1.70 (m, 1H), 1.39 (d, J=26.1 Hz, 9H), 1.19 (s,3H).

Example 67. Synthesis of Compound 171

To a solution of compound 170 (6.18 g, 15.0 mmol, 1.0 eq) in MeOH (50mL) was added Pd/C (0.6 g, 10% Pd/C) in a hydrogenation bottle. Themixture was shaken under 1 atm hydrogen atmosphere overnight, thenfiltered. The filtrate was concentrated to give the title compound as acolorless oil (4.8 g, 99% yield). ESI m/z: calcd for C₁₇H₂₆N₁O₅ [M+H]⁺:324.2, found 324.2o ¹H NMR (400 MHz, CDCl₃) δ 6.97 (d, J=6.5 Hz, 2H),6.74 (d, J=8.2 Hz, 2H), 3.93-3.66 (m, 1H), 2.58 (tdd, J=19.5, 12.9, 7.4Hz, 3H), 1.75 (ddd, J=20.1, 16.3, 7.7 Hz, 1H), 1.37 (d, J=21.5 Hz, 9H),1.11 (d, J=7.0 Hz, 3H).

Example 68. Synthesis of Compound 172

To a solution of compound 171(4.8 g, 15.0 mmol, 1.0 eq) in anhydrous THF(75 mL) was added slowly t-BuONO (18.0 mL, 150 mmol, 10.0 eq) at 0° C.under N₂, and the mixture was stirred at the same temperature for 3hours. After the consumption of starting material monitored by TLC, 1NKHSO₄ was added slowly to the mixture until pH 4. The resulting aqueoussolution was extracted with EA (150 mL, 75 mL×2), and the organicextract was washed with brine, dried over anhydrous sodium sulfate,filtered, concentrated in vacuo and the residue was purified by SiO₂column chromatography (PE:EA=3:1) to give the title compound as a yellowsolid (3.6 g, 65.4%). ESI m/z: calcd for C₁₇H₂₅N₂O₇ [M+H]⁺: 369.2, found369.2. ¹H NMR (400 MHz, MeOD) δ 7.93 (d, J=2.0 Hz, 1H), 7.48 (dd, J=8.6,2.1 Hz, 1H), 7.06 (d, J=8.5 Hz, 1H), 3.83-3.71 (m, 1H), 2.82 (dd,J=13.6, 5.0 Hz, 1H), 2.66-2.41 (m, 2H), 1.84 (ddd, J=14.0, 10.6, 5.6 Hz,1H), 1.65-1.51 (m, 1H), 1.28 (d, J=24.9 Hz, 9H), 1.15 (d, J=7.0 Hz, 3H).

Example 69. Synthesis of Compound 173

To a solution of compound 172 (3.2 g, 7.74 mmol, 1.0 eq) in MeOH (20 mL)was added Pd/C (0.2 g, 10% Pd/C) in a hydrogenation bottle. The mixturewas shaken under 1 atm H₂ atmosphere for 3 h. After consumption ofstarting material monitored by TLC, the mixture was filtered and thefiltrate was concentrated to give the title compound as a white foam(2.3 g, 92.0% yield). ESI m/z: calcd for C₁₇H₂₇N₂O₅ [M+H]⁺: 339.2, found339.2. ¹H NMR (400 MHz, MeOD) δ 6.61 (d, J=8.0 Hz, 2H), 6.45 (d, J=6.3Hz, 1H), 3.72 (d, J=7.3 Hz, 1H), 2.68-2.34 (m, 3H), 1.81-1.66 (m, 1H),1.56-1.45 (m, 1H), 1.36 (d, J=29.0 Hz, 9H), 1.08 (d, J=6.9 Hz, 3H).

Example 70. Synthesis of Compound 187

To a solution of compound 102 (1.00 g, 2.52 mmol) in acetonitrile (10mL) was added CCl₄ (2.2 mL, 22.7 mmol, 9.0 eq.) at −25° C. Afterstirring for 10 min, diisopropylethylamine (0.88 mL, 5.04 mmol, 2.0 eq.)and DMAP (0.03 g, 0.252 mmol, 0.1 eq.) were added, followed by dibenzylphosphite (0.84 mL, 3.78 mmol, 1.5 eq.). The reaction mixture wasallowed to reach r.t. over 1.5 h, and then quenched by a solution ofKH₂PO₄ (0.5 M, 50 mL). The reaction mixture was extracted with EtOAc(3×50 mL). The combined organic extracts were dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The residue was purifiedby flash column chromatography (10-50% EtOAc/PE) to afford compound 187(1.60 g, 96% yield) as a colorless oil.MS ESI m/z calcd for C₃₃H₄₁N₂O₁₀P[M+H]⁺ 657, found 657 Example 71. Synthesis of compound 188

To a solution of compound 187 (1.60 g, 2.43 mmol) in methanol (20 mL)was added Pd/C (10 wt %, 160 mg). The reaction mixture was stirred underH₂ atmosphere (1 atm) at r.t. for 3 h, then filtered through Celite andconcentrated under reduced pressure to afford compound 188 (1.00 g, 91%yield) as a white solid. MS ESI m/z calcd for C₁₉H₃₁N₂O₈P [M−H]⁻ 447,found 447.

Example 72. Synthesis of Compound 189

A solution of compound 188 (730 mg, 1.63 mmol) in ethanol (10 mL) wastreated with 1 N NaOH (16 mL, 16.3 mmol, 10 eq.) at r.t. overnight, andthen concentrated under reduced pressure. The residue was taken up inwater (20 mL) and acidified to pH 6 by 1 N HCl. The aqueous solution wasconcentrated under reduced pressure and the residue was triturated withMeOH/EtOAc (80:20, 5 mL), compound 189(0.68 g, 99% yield) was collectedfrom filtration as a white solid. MS ESI m/z calcd for C₁₇H₂₇N₂O₈P[M−H]⁻ 417, found 417.

Example 73. Synthesis of Compound 299

2-(2-aminoethoxy)ethanol (21.00 g, 200 mmol, 1.0 eq.) and K₂CO₃(83.00 g,600 mmol, 3.0 eq.) in acetonitrile (350 mL) was added BnBr (57.0 mL, 480mmol, 2.4 eq.). The mixture was refluxed overnight. Water (1 L) wasadded and extracted with EtOAc (3×300 mL). The combined organic layerswere washed with brine (1000 mL), dried over anhydrous Na₂SO₄, filtered,concentrated and purified by SiO₂ column chromatography (4:1hexanes/EtOAc) to give a colorless oil (50.97 g, 89.2% yield).MS ESI m/zcalcd for C₁₈H₂₃NO₂Na [M+Na]⁺309.17, found 309.19.

Example 74. Synthesis of Compound 300

To a mixture of 2-(2-(dibenzylamino)ethoxy)ethanol (47.17 g, 165.3 mmol,1.0 eq.), tert-butyl acrylate (72.0 mL, 495.9 mmol, 3.0 eq.) and n-Bu₄NI(6.10 g, 16.53 mmol, 0.1 eq.) in DCM (560 mL) was added sodium hydroxidesolution (300 mL, 50%). The mixture was stirred overnight. The organiclayer was separated and the water layer was extracted with EtOAc (3×100mL). The organic layers were washed with water(3×300 mL) and brine (300mL), dried over anhydrous Na₂SO₄, filtered, concentrated and purified bySiO₂ column chromatography (7:1 hexanes/EtOAc) to give a colorless oil(61.08 g, 89.4% yield).MS ESI m/z calcd for C₂₅H₃₆NO₄ [M+H]⁺ 414.2566,found 414.2384.

Example 75. Synthesis of Compound 301

To a solution of tert-butyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanoate (20.00 g, 48.36 mmol, 1.0 eq.) in THF (30 mL) and MeOH (60mL) was added Pd/C (2.00 g, 10 wt %, 50% wet) in a hydrogenation bottle.The mixture was shaken overnight, filtered through Celite (filter aid),and the filtrate was concentrated to afford a colorless oil (10.58 g,93.8% yield). MS ESI m/z calcd for C₁₁H₂₄NO₄ [M+H]⁺ 234.1627, found234.1810.

Example 76. Synthesis of Compound 302

To a solution of (E)-3-bromoacrylic acid(0.15 g, 1 mmol), DMAP (0.15 g,1.2 mmol) and DCC (0.21 g, 1 mmol) in DCM (10 ml), compound 301 (0.23 g,1 mmol) were added at 0° C. The reaction mixture was allowed to warm tor.t. and stirred overnight. The crude product was concentrated andpurified by SiO₂ column chromatography with a gradient of EA/DCM to givethe title product 302 (0.31 g, 85% yield). ESI MS m/z: calcd forC₁₄H₂₅BrNO₅ [M+H]⁺: 366.08, found 366.08.

Example 77. Synthesis of Compound 303

Compound 302 (0.31 g, 0.84 mmol) was dissolved in formic acid (4 mL) at0° C. then H₂O (2 mL) was added. The reaction mixture was allowed towarm to r.t. and stirred overnight. The crude product was concentratedand used for the next step without further purification. ESI MS m/z:calcd for C₁₀H₁₇BrNO₅ [M+H]⁺: 310.02, found 310.03.

Example 78. Synthesis of Compound 304

Compound 303 (0.12 g, 0.39 mmol), NHS (0.067 g, 0.58 mmol) and EDCI(0.11 g, 0.58 mmol) were dissolved in DCM (10 mL) and the mixture wasstirred at r.t. overnight, concentrated and purified by SiO₂ columnchromatography to give the title product 304 (0.13 g, 82% yield). ESI MSm/z: calcd for C₁₄H₂OBrN₂O₇ [M+H]⁺:407.04, found 407.04.

Example 79. Synthesis of Compound 326

A solution of 4-aminobutyric acid (7.5 g, 75 mmol) and NaOH (6 g, 150mmol) in H₂O (40 mL) was cooled to 0° C. and treated with a solution ofCbzCl (16.1 g, 95 mmol) in THF (32 ml) dropwise. After 1 h, the reactionwas allowed to warm to r.t. and stirred for 3 h. THF was removed undervacuum, the pH of the aqueous solution was adjusted to 1.5 by additionof 6 N HCl. The solution was extracted with ethyl acetate, and theorganic layer was washed with brine, dried and concentrated to givecompound 326 (16.4 g, 92% yield). MS ESI m/z calcd for C₁₂H₁₆NO₅ [M+H]⁺238.10, found 238.08.

Example 80. Synthesis of Compound 327

DMAP (0.8 g, 6.56 mmol) and DCC (17.1 g, 83 mmol) were added to asolution of 4-(((benzyloxy)carbonyl)amino)butanoic acid (16.4 g, 69.2mmol) and t-BuOH (15.4 g, 208 mmol) in DCM (100 mL). After stirring atr.t. overnight, the reaction was filtered and filtrate concentrated. Theresidue was dissolved in ethyl acetate and the washed with 1N HCl, brineand dried over Na₂SO₄. Concentration and purification by columnchromatography (10 to 50% EtOAc/hexanes) yielded compound 327 (7.5 g,37% yield). MS ESI m/z calcd for C₁₆H₂₃NO₄Na [M+Na]⁺316.16, found316.13.

Example 81. Synthesis of Compound 328

tert-Butyl 4-(((benzyloxy)carbonyl)amino)butanoate (560 mg, 1.91 mmol)was dissolved in MeOH (50 mL), and mixed with Pd/C catalyst (10 wt %,100 mg) then hydrogenated (1 atm) at r.t. for 3 h. The catalyst wasfiltered off and all volatiles were removed under vacuum to affordcompound 328 (272 mg, 90% yield). MS ESI m/z calcd for C₈H₁₈NO₂ [M+H]⁺160.13, found 160.13.

Example 82. Synthesis of Compound 330

tert-Butyl 4-aminobutanoate (477 mg, 3 mmol) and 2,3-dibromosuccinicacid (414 mg, 1.5 mmol) was dissolved in DCM (35 mL), to which DIPEA(1.16 g, 9 mmol) and EDC (0.86 g, 4.5 mmol) were added. The resultingsolution was stirred at r.t. overnight and then washed with brine, driedover Na₂SO₄. Filtration, concentration and purification by columnchromatography (pure DCM to 10% MeOH/DCM) yielded compound 330 (160 mg,22% yield). MS ESI m/z calcd for C₂₀H₃₄BrN₂O₆ [M+H]⁺ 477.15, found477.16.

Example 83. Synthesis of Compound 331

Compound 330 (80 mg, 0.168 mmol) was dissolved in DCM (5 mL) and treatedwith formic acid (8 mL) at 38° C. overnight. All volatiles were removedunder vacuum to afford compound 331 (61 mg, 99% yield). MS ESI m/z calcdfor C₁₂H₁₈BrN₂O₆ [M+H]⁺ 365.03, found 365.05.

Example 84. Synthesis of Compound 332

NHS (60 mg, 0.504 mmol) and EDCI (97 mg, 0.504 mmol) were added to asolution of compound 331 (61 mg, 0.168 mmol) in DCM (10 mL). Afterstirring at r.t. overnight, the reaction mixture was concentrated andpurified by column chromatography (0 to 10% MeOH/DCM) to afford compound332 (72 mg, 77% yield). MS ESI m/z calcd for C₂₀H₂₄BrN₄O₁₀ [M+H]⁺559.06, found 559.78.

Example 85. Synthesis of Compound 333

NaH₂PO₄ (0.1M in water, 1 mL) was added to a solution of compound 332(36 mg, 0.065 mmol) and compound 110 (25 mg, 0.063 mmol) in EtOH (5 mL).The resulting solution was stirred at r.t. overnight and thenHO—(PEG)24—NH₂ (95 mg) was added to the mixture and stirred at r.t.overnight. All volatiles were removed under vacuum and the residue waspurified by column chromatography (0 to 10% MeOH/DCM) to yield compound333 (28 mg, 24% yield). MS ESI m/z 1798.93 ([M+H]⁺).

Example 86. Synthesis of Compound 335

Compound 333 (28 mg, 0.0156 mmol) was dissolved in DCM (2 mL) andtreated with TFA (2 mL) at r.t. for 2 h. All volatiles were removedunder vacuum to afford compound 335 (25 mg, 98% yield), which was usedirectly in the next step. MS ESI m/z 1642.82 ([M+H]⁺).

Example 87. Synthesis of Compound 337

Compound 335 (25 mg, 0.0152 mmol) and perfluorophenyl ester 33a (15 mg,0.0213 mmol) were dissolved in DMA (5 mL). To the mixture, DIPEA (10 mg,0.077 mmol) was added. The resulting mixture was stirred at r.t.overnight, concentrated and purified by preparative HPLC (C₁₈ column,10-90% MeCN/H₂O) to afford compound 337 (13 mg, 40% yield).MS ESI m/z2163.82 ([M+H]⁺).

Example 88. Synthesis of Compound 341

To a solution of 2,2′-(ethane-1,2-diylbis(oxy))diethanol (55.0 mL,410.75 mmol, 3.0 eq.) in anhydrous THF (200 mL) was added sodium (0.1g). The mixture was stirred until Na disappeared and then tert-butylacrylate (20.0 mL, 137.79 mmol, 1.0 eq.) was added dropwise. The mixturewas stirred overnight and then quenched by HCl solution (20.0 mL, 1N) at0° C. THF was removed by rotary evaporation, brine (300 mL) was addedand the resulting mixture was extracted with EtOAc (3×100 mL). Theorganic layers were washed with brine (3×300 mL), dried over anhydrousNa₂SO₄, filtered and concentrated to afford a colorless oil (30.20 g,79.0% yield), which was used without further purification. MS ESI m/zcalcd for C₁₃H₂₇O₆ [M+H]⁺ 278.1729, found 278.1730.

Example 89. Synthesis of Compound 342

To a solution of tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propanoate (30.20 g, 108.5 mmol, 1.0 eq.) and TsCl (41.37 g, 217.0 mmol,2.0 eq.) in anhydrous DCM (220 mL) at 0° C. was added TEA (30.0 mL,217.0 mmol, 2.0 eq.). The mixture was stirred at room temperatureovernight, and then washed with water (3×300 mL) and brine (300 mL),dried over anhydrous Na₂SO₄, filtered, concentrated and purified by SiO₂column chromatography (3:1 hexanes/EtOAc) to give a colorless oil (39.4g, 84.0% yield).MS ESI m/z calcd for C₂₀H₃₃O₈S [M+H]⁺ 433.1818, found433.2838.

Example 90. Synthesis of Compound 343

To a solution of tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate (39.4 g, 91.1 mmol, 1.0 eq.) in anhydrous DMF(100 mL) wasadded NaN₃ (20.67 g, 316.6 mmol, 3.5 eq.). The mixture was stirred atroom temperature overnight. Water (500 mL) was added and extracted withEtOAc (3×300 mL). The combined organic layers were washed with water(3×900 mL) and brine (900 mL), dried over anhydrous Na₂SO₄, filtered,concentrated and purified by SiO₂ column chromatography (5:1hexanes/EtOAc) to give a light yellow oil (23.8 g, 85.53% yield).MS ESIm/z calcd for C₁₃H₂₅O₃N₅Na [M+Na]⁺326.2, found 326.2.

Example 91. Synthesis of Compound 344

Raney-Ni (7.5 g, suspended in water) was washed with water (three times)and isopropyl alcohol (three times) and mixed with compound 343 (5.0 g,16.5 mmol) in isopropyl alcohol. The mixture was stirred under a H₂balloon at r.t. for 16 h and then filtered over a Celite pad, withwashing of the pad with isopropyl alcohol. The filtrate was concentratedand purified by column chromatography (5-25% MeOH/DCM) to give a lightyellow oil (2.60 g, 57% yield). MS ESI m/z calcd for C₁₃H₂₈NO₅ [M+H]⁺279.19; found 279.19.

Example 92. Synthesis of Compound 345

Acetylenedicarboxylic acid (0.35 g, 3.09 mmol, 1.0 eq.) was dissolved inNMP (10 mL) and cooled to 0° C., to which compound 344 (2.06 g, 7.43mmol, 2.4 eq.) was added, followed by DMTMM (2.39 g, 8.65 mmol, 2.8 eq.)in portions. The reaction was stirred at 0° C. for 6 h and then dilutedwith ethyl acetate and washed with water and brine. The organic solutionwas concentrated and triturated with a mixture solvent of ethyl acetateand petroleum ether. The solid was filtered off and the filtrate wasconcentrated and purified by column chromatography (80-90% EA/PE) togive a light yellow oil (2.26 g, >100% yield), which was used withoutfurther purification. MS ESI m/z calcd for C₃₀H₅₃N₂O₁₂ [M+H]⁺ 633.35;found 633.30.

Example 93. Synthesis of Compound 346

Compound 345 (2.26 g) was dissolved in dichloromethane (15 mL) andcooled to 0° C. then treated with TFA (15 mL). The reaction was warmedto r.t. and stirred for 45 min, and then the solvent and residual TFAwas removed on rotovap. The crude product was purified by columnchromatography (0-15% MeOH/DCM) to give a light yellow oil (1.39 g, 86%yield for two steps). MS ESI m/z calcd for C₂₂H₃₇N₂O₁₂ [M+H]⁺ 521.23;found 521.24.

Example 94. Synthesis of Compound 380

Compound 110 (68 mg, 0.17 mmol), compound 124 (94.5 mg, 0.52 mmol) andHATU (162 mg, 0.425 mmol) were dissolved in DCM (50 mL). TEA (73 ul,0.52 mmol) was then added. The reaction mixture was stirred at r.t.overnight. Then the solvent was removed under reduced pressure and theresidue was purified by SiO₂ column to give the title product 380 (98mg, 80% yield). ESI m/z calcd for C₃₇H₄₉N₄O₁₁ [M+H]⁺: 725.33, found725.34.

Example 95. Synthesis of Compound 381

Compound 380 (98 mg, 0.135 mmol) dissolved in DCM (1.0 mL) was treatedwith TFA (1.0 mL) at r.t. for 2h, then concentrated and redissolved inDMA (1 mL), to which pentafluorophenyl ester 41a (44 mg, 0.06 mmol) andDIPEA (45.8 μL, 0.27 mmol) were added. The reaction was stirredovernight and then concentrated. The residue was purified by prep-HPLCwith a gradient of MeCN/H₂O to give the title product 381 (37 mg, 55%yield). ESI m/z calcd for C₅₃H₇₃N₈O₁₄S [M+H]⁺: 1077.49, found 1077.50.

Example 96. Synthesis of Compound 384

To a solution of (S)—2-amino-3-(4-nitrophenyl)propanoic acid (13.2 g,62.8 mmol) in methanol (120 mL) was added thionyl chloride (9 mL, 125.6mmol) at 0° C. The reaction mixture was heated to reflux and stirred for1 h, then concentrated under vacuum and suspended in ethyl acetate (50mL). The mixture was then filtered to afford the title compound as awhite solid (14.5 g, 91% yield). ESI m/z calcd for C₁₀H₁₃N₂O₄ [M+H]⁺:225.08, found 225.08.

Example 97. Synthesis of Compound 385

To a solution of compound 384 (9.5 g, 36.4 mmol) in THF (200 mL) wasadded triethylamine (12.6 mL, 91.1 mmol). After the mixture was stirredfor 30 minutes, di-tert-butyl dicarbonate (12.5 mL, 54.7 mmol) wasadded, and the reaction mixture was stirred for 1 h, then diluted withethyl acetate (200 mL), washed with 1 N HCl (30 mL), water (30 mL),dried over sodium sulfate, filtered and concentrated under vacuum toafford the title compound as a white solid (11.4 g, 97% yield). ESI m/zcalcd for C₅H₂₁N₂O₆ [M+H]⁺: 325.13, found 325.13.

Example 98. Synthesis of Compound 386

To a solution of compound 385 (14 g, 43.2 mmol) in anhydrousdichloromethane (150 mL) was added DIBAL-H (108 mL, 108 mmol) at −78° C.The reaction mixture was stirred at −78° C. for 30 min., then pouredinto ice water (200 mL), extracted with ethyl acetate (3×80 mL). Thecombined organic phase was washed with 1N HCl(2×50 mL), water (50 mL),dried over sodium sulfate, filtered, concentrated under vacuum, andpurified by silica gel column chromatography to afford the titlecompound (8.6 g, 68% yield). ESI m/z calcd for C₁₄H₁₉N₂O₅ [M+H]⁺:295.12, found 295.12.

Example 99. Synthesis of Compound 387

To a solution of compound 106 (8.1 g, 20.8 mmol) in DCM (100 mL) wasadded compound 386 (5.2 g, 17.8 mmol) at 0° C. The reaction mixture waswarmed to r.t. and stirred for 30 min. then concentrated under vacuumand purified by silica gel column to afford the title compound as ayellow solid (5.9 g, 82% yield). ESI m/z calcd for C₂₁H₃₁N₂O₆ [M+H]⁺:406.21, found 406.21.

Example 100. Synthesis of Compound 388

To a solution of compound 387 (4 g, 9.85 mmol) in MeOH (40 mL) was addedPd/C (0.4 g, 10 wt %) in a hydrogenation bottle. The mixture was stirredunder 1 atm H₂ overnight, filtered through Celite (filter aid), and thefiltrate was concentrated to afford compound 388 (3.6 g, yield˜100%).ESI m/z: calcd for C₂₁H₃₅N₂O₄ [M+H]⁺: 379.25, found 379.25.

Example 101. Synthesis of Compound 389

To a solution of compound 388 (3.6 g, 9.52 mmol) and triethylamine (1.3mL, 9.52 mmol) in dichloromethane (50 mL) was added4-nitrobenzenesulfonyl chloride (2.1 g, 9.52 mmol) at 0° C. The reactionmixture was warmed to r.t. and stirred for 1 h, then diluted with DCM(50 mL), washed with 1N HCl (20 mL), water (20 mL), dried over sodiumsulfate, filtered and concentrated under vacuum, then purified by silicagel column chromatography to afford the title compound as a yellow solid(4 g, 75% yield). ESI m/z calcd for C₂₇H₃₈N₃O₈S [M+H]⁺: 564.23, found564.23.

Example 102. Synthesis of Compound 390

To a solution of compound 389 (3.6 g, 6.39 mmol) in acetonitrile (40 mL)was added tert-butyl nitrite (2.29 mL, 19.1 mmol). The reaction mixturewas warmed to 45° C. and stirred for 6 hours. The reaction was thenconcentrated under vacuum and purified by silica gel columnchromatography to afford the title compound (3 g, 79% yield).ESI m/zcalcd for C₂₇H₃₇N₄O₁₀S [M+H]⁺: 609.22, found 609.22.

Example 103. Synthesis of Compound 391

To a solution of compound 390 (3.0 g, 4.92 mmol) in acetonitrile/DMSO(30 mL/1 mL) were added 4-methoxy thiophenol (2.76 g, 19.7 mmol) andpotassium carbonate (2.7 g, 19.7 mmol). The reaction mixture was stirredat the room temperature overnight, then diluted with ethyl acetate (100mL), washed with water (20 mL), brine (20 mL), dried over sodiumsulfate, filtered and concentrated under vacuum, and purified by silicagel column chromatography to afford the title compound (1.7 g, 85%yield). ESI m/z calcd for C₂₁H₃₄N₃O₆ [M+H]⁺: 424.24, found 424.24.

Example 104. Synthesis of Compound 392

To a solution of compound 391 (100 mg, 0.236 mmol) in MeOH (4 mL) wasadded Pd/C (10 mg, 10 wt %) in a hydrogenation bottle. The mixture wasstirred under 1 atm H₂ overnight, filtered through Celite (filter aid),and the filtrate was concentrated to afford the title compound (92.9 mg,˜100% yield). ESI m/z calcd for C₂₁H₃₆N₃O₄ [M+H]⁺: 394.26, found 394.26.

Example 105. Synthesis of Compound 393

Compound 392 (66 mg, 0.17 mmol), compound 124 (94.5 mg, 0.52 mmol) andHATU (162 mg, 0.425 mmol) were dissolved in DCM (50 mL). TEA (73 ul,0.52 mmol) was then added. The reaction mixture was stirred at r.t.overnight, the solvent was removed under reduced pressure and theresidue was purified by SiO₂ column to give the title product 393 (98mg, 80% yield). ESI m/z calcd for C₃₇H₅₀N₅O_(1O) [M+H]⁺: 724.35, found724.35.

Example 106. Synthesis of Compound 394

Compound 393 (98 mg, 0.135 mmol) dissolved in DCM (1.0 mL) was treatedwith TFA (1.0 mL) at r.t. for 2 h, then concentrated to give compound394, which was used in the next step without further purification.

Example 107. Synthesis of Compound 395

To a solution of compound 394 (76.9 mg, 0.135 mmol) in DMA (1 mL) wasadded pentafluorophenyl ester 41a (44 mg, 0.06 mmol) and DIPEA (45.8 μL,0.27 mmol). The reaction was stirred overnight, then concentrated andthe residue was purified by prep-HPLC with a gradient of MeCN/H₂O togive the title product 395 (37 mg, 55% yield). ESI m/z calcd forC₅₃H₇₄N₉O₁₃S [M+H]⁺: 1076.50, found 1076.50.

Example 108. Synthesis of Compound 409

To a solution of maleimide (6.35 g, 65.4 mmol, 1.0 eq.) in EtOAc (120mL) were added N-methyl morpholine (8.6 mL, 78.5 mmol, 1.2 eq.) andmethyl chloroformate (6.0 mL, 78.5 mmol, 1.2 eq.) at 0° C. The reactionwas stirred at 0° C. for 30 min and r.t. 1 h. The solid was filtered offand filtrate concentrated. The residue was dissolved in CH₂Cl₂ andfiltered through a silica gel plug and eluated with CH₂Cl₂ to remove thecolor. The appropriate fractions were concentrated and resulted solidwas triturated with 10% EtOAc/PE to give a white solid (9.00 g, 89%yield).

Example 109. Synthesis of Compound 410

A mixture of compound 301 (8.16 g, 35.0 mmol, 1.0 eq.) and saturatedNaHCO₃(40 mL) was cooled to 0° C., to which compound 409 (5.43 g, 35.0mmol, 1.0 eq.) was added in portions. After stirring at 0° C. for 1 h,the reaction was warmed to r.t. and stirred for 1 h. The reaction wasextracted with DCM (3×100 mL) and the organic extract was washed withbrine, dried over anhydrous Na₂SO₄, concentrated and purified by SiO₂column chromatography to give a white solid (6.76 g, 62% yield). MS ESIm/z calcd for C₁₅H₂₃NO₆ [M+H]⁺ 314.15, found 314.15.

Example 110. Synthesis of Compound 411

A solution of compound 410 (1.85 g, 5.9 mmol) was dissolved in DCM (20mL) and treated with TFA (7 mL) at r.t. for 16 h, then concentrated andpurified by SiO₂ column chromatography (11:1 DCM/MeOH) to give a whitefoam (1.47 g, 97% yield). MS ESI m/z calcd for C₁₁H₁₅NO₆ [M+H]⁺ 258.09,found 258.09.

Example 111. Synthesis of Compound 412

Compound 110 (100 mg, 0.25 mmol), compound 411 (65 mg, 0.25 mmol) andHATU (190 mg, 0.5 mmol) were dissolved in DCM (50 ml). TEA (73 μL, 0.5mmol) was added and the reaction mixture was stirred at r.t. overnight.Then the solvent was removed under reduced pressure and the residue waspurified by SiO₂ column to give the title product 412 (164 mg, 75%yield). ESI m/z calcd for C₄₃H₆₁N₄O₁₅ [M+H]⁺: 873.41, found 873.41.

Example 112. Synthesis of Compound 413

Compound 412 (52.4 mg, 0.06 mmol) dissolved in DCM (1.0 mL) was treatedwith TFA (1.0 mL) at r.t. for 2h, then concentrated and re-dissolved inDMA (1 mL), to which pentafluorophenyl ester 41a (44 mg, 0.06 mmol) andDIPEA (34 μL, 0.20 mmol) were added. The reaction was stirred overnightand then concentrated. The residue was purified by prep-HPLC with agradient of MeCN/H₂O to give the title product 413 (33 mg, 45% yield).ESI m/z calcd for C₅₉H₈₅N₈O₁₈S [M+H]⁺: 1225.56, found 1225.55.

Example 113. Synthesis of Compound 415

Compound 392 (98 mg, 0.25 mmol), compound 411 (130 mg, 0.5 mmol) andHATU (190 mg, 0.5 mmol) were dissolved in DCM (50 ml). TEA (73 μL, 0.5mmol) was added and the reaction mixture was stirred at r.t. overnight.The reaction solvent was removed under reduced pressure and the residuewas purified by SiO₂ column to give the title product 415 (163 mg, 75%yield). ESI m/z calcd for C₄₃H₆₂N₅O₁₄ [M+H]⁺: 872.42, found 872.42.

Example 114. Synthesis of Compound 416

Compound 415 (54.3 mg, 0.06 mmol) dissolved in DCM (1.0 mL) was treatedwith TFA (1.0 mL) at r.t. for 2 h, then concentrated and re-dissolved inDMA (1 mL), to which pentafluorophenyl ester 41a (44 mg, 0.06 mmol) andDIPEA (34 μL, 0.20 mmol) were added. The reaction was stirred overnight,then concentrated and the residue was purified by prep-HPLC with agradient of MeCN/H₂O to give the title product 416 (33 mg, 45% yield).ESI m/z calcd for C₅₉H₈₆N₉O₁₇S [M+H]⁺: 1224.58, found 1224.58.

Example 115. Synthesis of Compound 419

A mixture of N-Boc-ethylenediamine (5.6 mL, 35.4 mmol, 1.1 eq.) andsaturated NaHCO₃(60 mL) was cooled to 0° C., to which compound 409 (5.00g, 32.2 mmol, 1.0 eq.) was added in portions. After stirring at 0° C.for 30 min, the reaction was warmed to r.t. and stirred for 1 h. Theprecipitate was collected by filtration and washed with cold water, thendissolved in EtOAc and washed with brine, dried over anhydrous Na₂SO₄and concentrated to give a white solid (6.69 g, 87% yield).

Example 116. Synthesis of Compound 420

A solution of compound 419 (6.00 g, 25.0 mmol), furan (18.0 mL) intoluene (120 mL) in a high pressure tube was heated to reflux andstirred for 16 h. The colorless solution turned yellow during reaction.The mixture was then cooled to r.t. and concentrated. The resultingwhite solid was triturated with ethyl ether to give compound 420 (6.5 g,84% yield).

Example 117. Synthesis of Compound 421

A solution of compound 420 (9.93 g, 32.2 mmol) was dissolved in dioxane(15 mL) and treated with concentrated HCl (15 mL) at r.t. for 3 h. Thereaction was concentrated and the resulting solid was collected byfiltration, with washing of the filter cake with EtOAc. The solid wasdried in an oven (50° C.) overnight to give compound 421 (6.94 g, 88%yield).

Example 118. Synthesis of Compound 422

To a solution of compound 421 (0.85 g, 3.47 mmol) in THF (10 mL) wasadded POCl₃ (162 μL, 1.73 mmol) at −10° C., followed by TEA (966 μL,6.95 mmol). The reaction was stirred at −10° C. for 3h, and then thesolution was diluted with DCM (20 mL) and filtered over Celite, thefiltrate was concentrated to give compound 422, which was used in thenext step directly. ESI m/z calcd for C₂₀H₂₃ClN₄O₇P [M+H]⁺: 497.09,found 497.09.

Example 119. Synthesis of Compound 423

Compound 422 (0.50 g, 1.0 mmol) and DIPEA (0.4 mL, 2.4 mmol) weredissolved in DCM (5.0 mL) at 0° C., and then compound 301 (0.23 g, 1.0mmol) was added. The reaction was stirred at 0° C. for 2.5h, thenconcentrated and purified by SiO₂ column to give the title product 423(0.30 g, 43%). ESI m/z calcd for C₃₁H₄₅N₅O₁₁P [M+H]⁺: 694.28, found694.28.

Example 120. Synthesis of Compound 424

Compound 423 (0.30 g, 0.5 mmol) was dissolved in DCM (3 mL), and treatedwith TFA (3 mL) at r.t. for 2h, then concentrated to give compound 424,which was used in the next step without further purification.

Example 121. Synthesis of Compound 425

Compound 424 (40 mg, 0.063 mmol), compound 110 (40 mg, 0.10 mmol), HATU(24 mg, 0.063 mmol) were dissolved in DCM (5 mL), and then TEA (27.8 μL,0.2 mmol) was added. The reaction mixture was stirred at r.t. overnight.Then the solvent was removed under reduced pressure and the residue waspurified by SiO₂ column to give the title product 425 (53.4 mg, 84%yield). ESI m/z calcd for C₄₈H₆₉N₇O₁₅P [M+H]⁺: 1014.45, found 1014.45.

Example 122. Synthesis of Compound 426

Compound 425 (53.4 mg, 0.053 mmol) was dissolved in DCM (2 mL), andtreated with TFA (2 mL) at r.t. for 2 h, then concentrated to givecompound 426, which was used in the next step without furtherpurification.

Example 123. Synthesis of Compound 427

To a solution of compound 426 (45.0 mg, 0.053 mmol) in DMA (1 mL) wereadded pentafluorophenyl ester 41a (37.0 mg, 0.053 mmol) and DIPEA (17μL, 0.1 mmol). The reaction was stirred overnight and concentrated. Theresidue was purified by prep-HPLC with a gradient of MeCN/H₂O to givethe title product 427 (26.2 mg, 36% yield). ESI m/z calcd forC₆₄H₉₃N₁₁O₁₈PS [M+H]⁺: 1366.61, found 1366.61.

Example 124. Synthesis of Compound 428

Compound 427 (8.0 mg, 0.0058 mmol) was dissolved in toluene (5.0 mL) andheated to reflux overnight, then concentrated and purified by prep-HPLCwith a gradient of MeCN/H₂O to give the title product 428 (6.4 mg, 90%yield). ESI m/z calcd for C₅₆H₈₅N₁₁O₁₆PS [M+H]⁺: 1230.56, found 1230.56.

Example 125. Synthesis of Compound 432

NaH (60%, 8.0 g, 200 mmol) was added to a solution of2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-ol (42.8 g, 100 mmol) in THF(1.0 L). After stirring at r.t. for 30 min, tert-butyl 2-bromoacetate(48.8 g, 250 mmol) was added to the mixture, and stirred at r.t. for 1h. The mixture was then poured onto ice water, extracted with DCM, andthe organic layer was washed with brine, dried over anhydrous Na₂SO₄.Purification by column chromatography (0% to 5% MeOH: DCM) yieldedcompound 432 as a yellow oil(32 g, 59% yield).

Example 126. Synthesis of Compound 433

Compound 432 (40.0 g, 73.8 mmol) was dissolved in DCM (400 mL), and thenformic acid (600 mL) was added. The resulting solution was stirred at25° C. overnight. All volatiles were removed under vacuum, whichafforded the title product as yellow oil (36.0 g, theoretical yield).ESI m/z calcd for C₂₁H₄₃O₁₂ [M+H]⁺: 487.27, found 487.24.

Example 127. Synthesis of Compound 434

To the solution of compound 433 (36.0 g, 73.8 mmol) dissolved in DCM(640 mL), (COCl)₂ (100 mL) and DMF (52 g, 0.74 mmol) were added. Theresulting solution was stirred at r.t. for 4 h. All volatiles wereremoved under vacuum to yield the title product as a yellow oil.

Example 128. Synthesis of Compound 436

Z-L-Lys-OH (41.4 g, 147.6 mmol), Na₂CO₃ (23.4 g, 221.4 mmol) and NaOH(5.9 g, 147.6 mmol) were dissolved in water (720 mL). The mixture wascooled to 0° C., to which a solution of compound 434 (37.2 g, 73.8 mmol)in THF (20 mL) was added. The resulting mixture was stirred at r.t. for1 h. THF was removed under vacuum, and concentrated HCl was added to theaqueous solution until pH reached 3 under ice cooling. After extractionwith DCM, the organic layer was washed with brine, dried over Na₂SO₄ andconcentrated to give the title product as yellow oil (55 g, 99% yield).ESI m/z calcd for C₃₅H₆₀N₂O₁₅ [M+H]⁺: 749.40, found 749.39.

Example 129. Synthesis of Compound 437

HATU (39.9 g, 105 mmol) was added to a solution of4-(((benzyloxy)carbonyl)amino) butanoic acid (26.1 g, 110 mmol) in DMF(300 mL). After stirring at r.t. for 30 min, the mixture was added to asolution of compound 110 (39.4 g, 100 mmol) and TEA (20.2 g, 200 mmol)in DMF (300 mL). The resulting mixture was stirred at r.t. for 2 h.Water was then added, extracted with EtOAc, the organic layer was washedwith brine, dried over Na₂SO₄. Purification by column chromatography(20% to 70% EA/PE) yielded the title product as a white solid (45 g, 73%yield). ESI m/z calcd for C₃₃H₄₈N₃O₈ [M+H]⁺: 614.34, found 614.15.

Example 130. Synthesis of Compound 438

Compound 437 (100 g, 163 mmol) was dissolved in methanol (500 mL) andhydrogenated (1 atm) with Pd/C catalyst (10 wt %, 10 g) at r.t.overnight. The catalyst was filtered off and the filtrate wereconcentrated under reduced pressure to afford compound 438 (75.8 g, 97%yield) as a brown foamy solid. ¹H NMR (400 MHz, CDCl₃) δ 7.11 (s, 1H),6.83 (d, J=10.3 Hz, 2H), 5.04-4.52 (m, 6H), 3.90-3.56 (m, 1H), 2.81 (d,J=5.3 Hz, 2H), 2.63 (dd, J=12.5, 6.1 Hz, 2H), 2.54-2.26 (dd, J=14.0, 7.6Hz, 4H), 1.94-1.64 (m, 3H), 1.44-1.36 (m, 18H), 1.08 (d, J=6.9 Hz, 3H).ESI m/z calcd for C₂₅H₄₂N₃O₆ [M+H]⁺: 480.30, found 480.59.

Example 131. Synthesis of Compound 439

To a solution of compound 436 (130 g, 174 mmol, 1.1 eq.) in DMF (500 mL)were added TEA (66 mL, 474 mmol, 3 eq.) and HATU (72 g, 190 mmol, 1.2eq.) in sequence at 0° C. Then the reaction mixture was warmed to r.tand stirred for 2 h. A solution of compound 438 (75.8 g, 158 mmol, 1.0eq) in DMF (500 mL) was added to the above solution at 0° C., and thereaction mixture was stirred at r.t. for 1 h. The reaction mixture waspoured into water (4 L), the aqueous layer was extracted with EtOAc(3×500 mL), and the organic layers were combined and washed with brine(2 L), dried over Na₂SO₄, concentrated and the crude product 439 (190 g)was used in the next step directly. ESI: m/z: calcd for C₆H₁₀₀N₅O₂O[M+H]⁺: 1210.69, found 1210.69.

Example 132. Synthesis of Compound 440

The crude product from previous reaction 439 (190 g) was dissolved inmethanol (900 mL) and hydrogenated (1 atm) with Pd/C catalyst (10 wt %,19 g) at r.t. overnight. The catalyst was filtered off and the filtratewere concentrated under reduced pressure, and the crude compound waspurified by SiO₂ column with a gradient of DCM/MeOH to give the titleproduct 440 (105 g, 62% yield over two steps) as a brown oil. ESI m/zcalcd for C₅₂H₉₅N₅O₁₈ [M+H]⁺: 1077.65, found 1077.65.

Example 133. Synthesis of Compound 441

To a solution of compound 440 (105 g, 97.1 mmol, 1.0 eq.) in EtOH (5.3L) was added compound 125 (54.4 g, 194.2 mmol, 2.0 eq) at r.t. Then 0.1MNaH₂PO₄ solution (1.1 L) was added, and the reaction mixture was stirredat r.t. overnight. EtOH was then evaporated under vacuum and the residuewas poured onto water (3 L). The aqueous solution was extracted withEtOAc (4×500 mL), the organic layers were combined and washed with brine(2 L), dried over Na₂SO₄, concentrated and the crude product waspurified by SiO₂ column with a gradient of DCM/MeOH to give the titlecompound 441 (100 g, 83% yield) as a yellow oil. ¹H NMR (400 MHz, CDCl₃)δ 9.43 (s, 1H), 7.35 (s, 1H), 7.23 (t, J=5.1 Hz, 1H), 7.01 (d, J=4.5 Hz,2H), 6.89 (s, 2H), 6.70 (s, 2H), 4.56-4.45 (m, 1H), 4.30 (t, J=9.7 Hz,1H), 3.97 (s, 2H), 3.86-3.74 (m, 1H), 3.66-3.63 (m, 36H), 3.58-3.52 (m,5H), 3.38 (s, 3H), 3.33-3.19 (m, 3H), 2.47 (d, J=6.2 Hz, 4H), 2.23 (dd,J=11.6, 6.1 Hz, 2H), 1.91 (dtd, J=26.8, 13.6, 6.5 Hz, 7H), 1.71 (d,J=7.7 Hz, 2H), 1.56-1.49 (m, 2H), 1.42 (s, 9H), 1.39 (s, 9H), 1.10 (d,J=6.5 Hz, 3H). ESI m/z calcd for C₆₀H₁₀₁N₆O₂₁ [M+H]⁺: 1241.69, found1241.69.

Example 134. Synthesis of Compound 442

A solution of compound 441 (79.1 mg, 0.062 mmol) in DCM (2 mL) wastreated with TFA (2 mL) at r.t. for 2 h then concentrated andco-evaporated with toluene to give a crude product 442, which was useddirectly in the next step.

Example 135. Synthesis of Compound 443

Compound 442 (67 mg, 0.062 mmol) and compound 41a (43 mg, 0.062 mmol)were dissolved in DMA (4 mL). And then DIPEA (43 μL, 0.248 mmol) wasadded. The resulting mixture was stirred at r.t. for 3 h. After thesolvent was removed under vacuum, the residue was purified onpreparative HPLC (C₁₈ column, 10-90% MeCN/H₂O) to afford the titleproduct 443 (59 mg, 60% yield). ESI m/z calcd for C₇₆H₁₂₅N₁₀O₂₄S [M+H]⁺:1594.92, found 1594.24.

Example 136. Synthesis of Compound 457

NaH (60%, 0.64 g, 16 mmol) was added to a solution of2,5,8,11,14,17-hexaoxanonadecan-19-ol (2.37 g, 8 mmol) in THF(25 mL).After stirring at r.t. for 15 min, tert-butyl 2-bromoacetate (3.90 g, 20mmol) was added and the reaction was stirred at r.t. overnight. Thereaction mixture was poured onto ice water, extracted with DCM. Theorganic layer was washed with brine, dried over Na₂SO₄, purified bycolumn chromatography (20% to 50% PE/EtOAc) to yield the title compound(1.47 g, 45%) as a colorless oil. ESI m/z calcd for C₁₉H₃₉O₉ [M+H]⁺:411.25, found 411.15.

Example 137. Synthesis of Compound 458

Compound 457 (1.47 g, 3.60 mmol) was dissolved in DCM (30 mL), andtreated with formic acid (50 mL). The resulting solution was stirred at38° C. overnight. All volatiles were removed under vacuum, whichafforded the title compound (1.20 g, 94% yield) as a yellow oil. ESI m/zcalcd for C₁₅H₃₁O₉ [M+H]⁺: 355.19, found 355.18.

Example 138. Synthesis of Compound 459

Compound 458 (1.10 g, 3.20 mmol) was dissolved in DCM (20 mL), to themixture, (COCl)₂ (4 mL) and DMF (3 drops) were added. The resultingsolution was stirred at r.t. for 4 h. All volatiles were removed invacuum to give the title compound as a yellow oil, which was useddirectly in the next step.

Example 139. Synthesis of Compound 460

Z-L-Lys-OH (1.80 g, 6.4 mmol), Na₂CO₃ (1.00 g, 9.6 mmol) and NaOH (0.26g, 6.4 mmol) were dissolved in water (30 mL) and cooled to 0° C., then asolution of compound 459 (1.20 g, 3.2 mmol) in THF (10 mL) was added.The resulting mixture was stirred at r.t. for 1 h. THF was removed undervacuum, and concentrated HCl was added to reach pH 3 under ice cooling.The solution was extracted with DCM, and the organic layer was washedwith brine, dried over Na₂SO₄, concentrated to give the title compound(1.77 g, 90%) as a brown oil. ESI m/z calcd for C₂₉H₄₉N₂O₁₂ [M+H]⁺:617.32, found 617.31.

Example 140. Synthesis of Compound 461

NHS (644 mg, 5.60 mmol) and EDC (1.08 g, 5.60 mmol) were added to asolution of compound 460 (2.30 g, 3.70 mmol) in DCM (100 mL). Afterstirring at r.t. overnight, the reaction mixture was loaded on silicagel column and purification by column chromatography (0% to 10%MeOH/DCM) yielded the title compound (2.10 g, 80% yield) as a brown oil.ESI m/z calcd for C₃₃H₅₂N₃O₁₄ [M+H]⁺: 714.34, found 714.32.

Example 141. Synthesis of Compound 462

NaH₂PO₄ (0.1M, 3 mL) was added to a solution of compound 461 (357 mg,0.50 mmol) and compound 110 (200 mg, 0.50 mmol) in EtOH (15 mL). Theresulting solution was stirred at r.t. for 24 h. All volatiles wereremoved under vacuum, and the residue was purified by columnchromatography (5% to 10% MeOH/DCM) to yield the title compound (216 mg,44% yield) as a brown oil. ESI m/z calcd for C₅₀H₈₁N₄O₁₆ [M+H]⁺: 993.56,found 993.57.

Example 142. Synthesis of Compound 463

Compound 462 (108 mg, 0.109 mmol) was dissolved in MeOH (5 mL) andstirred with palladium catalyst (10% on carbon, 50 mg) under hydrogenatmosphere (1 atm) at r.t. for 3 h. The catalyst was filtered off andall volatiles were removed under vacuum, which afforded the titlecompound (94 mg, theoretical yield) as a yellow oil. ESI m/z calcd forC₄₂H₇₅N₄O₁₄ [M+H]⁺: 859.52, found 859.93.

Example 143. Synthesis of Compound 464

NaH₂PO₄ (0.1M, 2.0 mL) was added to a solution of compound 463 (94 mg,0.109 mmol) and compound 125 (61 mg, 0.218 mmol) in EtOH (10 mL). Theresulting solution was stirred at r.t. for 24 h. All volatiles wereremoved under vacuum, and purification by column chromatography (5% to10% MeOH/DCM) yielded the title compound (40 mg, 36% yield) as a yellowoil. ESI m/z calcd for C₅₀H₈₂N₅O₁₇ [M+H]⁺: 1024.56, found 1024.98.

Example 144. Synthesis of Compound 465

Compound 464 (20 mg, 0.0196 mmol) was dissolved in DCM (3 mL) andtreated with TFA (3 mL) at r.t. for 2 h. All volatiles were removed invacuum, which afforded the title compound (17.0 mg, theoretical yield)as a yellow oil. ESI m/z calcd for C₄₁H₆₆N₅O₁₅ [M+H]⁺: 868.45, found868.47.

Example 145. Synthesis of Compound 466

Compound 465 (17.0 mg, 0.0196 mmol) and compound 41a (14 mg, 0.0196mmol) were dissolved in DMA (3 mL). To the mixture, DIPEA (10 μL, 0.0588mmol) was added. The resulting mixture was stirred at r.t. for 3 h. Thesolvent was then removed under vacuum, and the residue was purified onpreparative HPLC (C₁₈ column, 10-90% MeCN/H₂O) to afford the titlecompound 466 (15 mg, 64% yield) as a yellow oil. ESI m/z calcd forC₆₆H₁₀₆N₉O₂₀S [M+H]⁺: 1376.72, found 1376.72.

Example 146. Synthesis of Compound 487

Compound 110 (0.30 g, 0.76 mmol), compound Z-L-Ala-OH(0.17 g, 0.76 mmol)and HATU (0.29 g, 0.76 mmol) were dissolved in DCM (20 mL), to which TEA(110 μL, 0.8 mmol) was added. The reaction mixture was stirred at r.t.overnight. Then the solvent was removed under reduced pressure and theresidue was purified by SiO₂ column to give the title product 487 (0.43g, 95% yield). ESI m/z calcd for C₃₂H₄₆N₃O₈ [M+H]⁺: 600.32, found600.32.

Example 147. Synthesis of Compound 488

In a hydrogenation bottle, Pd/C (0.10 g, 33 wt %, 50% wet) was added toa solution of compound 487 (0.3 g, 0.5 mmol) in MeOH (10 mL). Themixture was shaken overnight under 1 atm H₂ then filtered through Celite(filter aid), the filtrate was concentrated and mixed with compound 461(357 mg, 0.5 mmol) in EtOH (20 mL). NaH₂PO₄ (0.1M, 4 mL) was added andthe resulting solution was stirred at r.t. for 24 h. All volatiles wereremoved under vacuum, and purification of the residue by columnchromatography (5% to 10% MeOH/DCM) yielded the title compound (176 mg,33%) as a yellow oil. ESI m/z calcd for C₅₃H₈₆N₅O₁₇ [M+H]⁺: 1064.59,found 1064.60.

Example 148. Synthesis of Compound 489

Compound 488 (176 mg, 0.166 mmol) was dissolved in MeOH (15 mL), and washydrogenated (1 atm) with palladium catalyst (10%, 80 mg) at r.t. for3h. The catalyst was filtered off and all volatiles were removed undervacuum, which afforded the title compound (154 mg, theoretical yield) asa yellow oil. ESI m/z calcd for C₄₅H₈₀N₅O₁₅ [M+H]⁺: 930.56, found930.56.

Example 149. Synthesis of Compound 490

NaH₂PO₄ (0.1 M, 4 ml) was added to a solution of compound 489 (154 mg,0.166 mmol) and compound 125 (93 mg, 0.332 mmol) in EtOH (20 mL). Theresulting solution was stirred at r.t. for 24 h. All volatiles wereremoved under vacuum, purification by column chromatography (5% to 10%MeOH: DCM) yielded the title compound (117 mg, 64%) as a yellow oil. ESIm/z calcd for C₅₃H₈₇N₆O₁₈ [M+H]⁺: 1095.60, found 1095.61.

Example 150. Synthesis of Compound 491

Compound 490 (39 mg, 0.0356 mmol) was dissolved in DCM (3 mL) andtreated with TFA (3 mL) at r.t. for 2 h. All volatiles were removedunder vacuum, which afforded the title compound (33 mg, theoreticalyield) as a yellow oil. ESI m/z calcd for C₄₄H₇₁N₆O₁₆ [M+H]⁺: 939.48,found 939.49.

Example 151. Synthesis of Compound 492

Compound 491 (33 mg, 0.0356 mmol) and compound 41a (25 mg, 0.0356 mmol)was dissolved in DMA (3 mL), to which DIPEA (15 mg, 0.116 mmol) wasadded. The resulting mixture was stirred at r.t. for 3 h. The solventwas removed under vacuum, and the residue was purified on preparativeHPLC (C₁₈ column, 10-90% o MeCN/H₂O) to afford the title compound 492(17 mg, 33% o) as a yellow oil. ESI m/z calcd for C₆₉H₁₁₁N₁₀O₂₁S [M+H]⁺:1447.76, found 1448.78.

Example 152. Synthesis of Compound 495

2-(Dimethylamino)acetic acid(0.60 g, 4.30 mmol) and HATU (1.08 g, 2.86mmol) were dissolved in DMF (2 mL), to which TEA (1 mL, 7.16 mmol) wasadded. After stirring at r.t. for 1 h, a solution of Z-L-Lys-OH (0.80 g,2.86 mmol) in DMF (2 mL) was added. The reaction mixture was stirred atr.t. for 2 h and then concentrated under reduced pressure. The residuewas purified by prep-HPLC with a gradient of MeCN/H₂O to give the titlecompound 495 (0.50 g, 50% yield) as a colorless oil. ESI m/z calcd forC₁₈H₂₈N₃O₅ [M+H]⁺:366.20, found 366.20.

Example 153. Synthesis of Compound 496

To a solution of carboxylic acid 495 (0.50 g, 1.37 mmol) in DCM (15 mL)were added pentafluorophenol (0.38 g, 2.05 mmol) and EDCI (0.52 g, 2.74mmol). The reaction mixture was stirred at r.t. overnight, and thenfiltered over Celite, with washing of the filter cake with DCM. Thefiltrate was concentrated and the resulting PFP-ester was dissolved in10 mL DCM. Compound 438 (0.44 g, 0.91 mmol) and i-Pr₂EtN (0.32 mL, 1.82mmol) were added and the reaction mixture was stirred at r.t. for 2 h,then concentrated. The residue was purified by SiO₂ column using agradient of MeOH/DCM to deliver product 496 (1.02 g, theoretical yield).ESI m/z calcd for C₄₃H₆₇N₆O₁₀ [M+H]⁺: 827.48, found 827.48.

Example 154. Synthesis of Compound 497

Compound 496 (1.02 g, 1.23 mmol) was dissolved in MeOH (10 mL) andstirred with palladium catalyst (10% on carbon, 100 mg) under hydrogenatmosphere (1 atm) at r.t. overnight. The catalyst was filtered off andall volatiles were removed under vacuum, which afforded the titlecompound (0.76 g, 89% yield). ESI m/z calcd for C₃₅H₆₁N₆O₈[M+H]⁺:693.45, found 693.45.

Example 155. Synthesis of Compound 498

NaH₂PO₄ (0.1M, 1 mL) was added to a solution of compound 497 (0.25 g,0.36 mmol, 1 eq) and compound 125 (0.15 g, 0.54 mmol, 1.5 eq) in EtOH (5mL). The resulting solution was stirred at r.t. overnight. All volatileswere removed under vacuum, and purification by column chromatography (5%to 10% MeOH/DCM) yielded the title compound (0.15 g, 48% yield). ESI m/zcalcd for C₄₃H₆₈N₇O₁₁ [M+H]⁺: 858.49, found 858.49.

Example 156. Synthesis of Compound 499

Compound 498 (0.15 g, 0.175 mmol) was dissolved in DCM (1 mL) andtreated with TFA (2 mL) at r.t. for 2 h. All volatiles were removed invacuum, and the residue was dissolved in DMA (2 mL), to whichpentafluorophenyl ester 41a (121.1 mg, 0.175 mmol) was added, followedby DIPEA (91 μL, 0.525 mmol). The reaction was stirred overnight andconcentrated, purified by prep-HPLC with a gradient of MeCN/H₂O to givethe title product 499 (30.7 mg, 14%). ESI m/z calcd for C₅₉H₉₂N₁₁O₁₄S[M+H]⁺: 1210.65, found 1210.62.

Example 157. Synthesis of Compound 501

To a solution of H-Lys-OH (0.31 g, 2.14 mmol, 1 eq) in EtOH (20 mL) wasadded compound 125 (1.80 g, 6.42 mmol, 3 eq) at r.t. Then 0.5 M Na₂HPO₄(4 mL) was added, and the reaction mixture was stirred at r.t.overnight. After solvents were evaporated under vacuum, the residue waspurified by prep-HPLC with a gradient of H₂O/MeCN to give the titlecompound 501 (0.26 g, 26%). ESI m/z calcd for C₂₂H₂₉N₄O₈ [M+H]⁺:477.19,found 477.19.

Example 158. Synthesis of Compound 502

To a solution of carboxylic acid 501 (0.26 g, 0.55 mmol) in DCM (10 mL)were added NHS (0.095 g, 0.825 mmol) and EDCI (0.16 g, 0.825 mmol). Thereaction mixture was stirred at r.t. overnight, then concentrated anddiluted with H₂O (50 mL), extracted with EtOAc (2×20 mL). Combinedorganic layers were dried over Na₂SO₄, filtered and concentrated to givea crude product 502 (0.34 g), which was used in the next step directly.ESI m/z calcd for C₂₆H₃₂N₅O₁₀ [M+H]⁺:574.21, found 574.21.

Example 159. Synthesis of Compound 503

To a solution of compound 438 (0.19 g, 0.4 mmol, 1.0 eq.) in EtOH (30mL) were added compound 502 (0.34 g, 0.6 mmol, 1.5 eq.) and 0.1 MNaH₂PO₄ (6 mL). The reaction mixture was stirred at r.t. overnight andthen concentrated under vacuum. The residue was diluted with H₂O (100mL), extracted with EtOAc (2×40 mL). The combined organic layers weredried over Na₂SO₄, then purified by SiO₂ column with a gradient ofDCM/MeOH to give the title product 503 (0.115 g, 31%). ESI m/z calcd forC₄₇H₆₈N₇O₁₃ [M+H]⁺: 938.48, found 938.49.

Example 160. Synthesis of Compound 504

To a solution of compound 503 (0.115 g, 0.12 mmol) in 1 mL of DCM wasadded 2 mL of TFA, and the reaction mixture was stirred at r.t. for 2h,then concentrated and purified by prep-HPLC with a gradient of H₂O/MeCNto give the title compound 504 (0.0312 g, 33%). ESI m/z calcd forC₂₂H₂₉N₄O₈ [M+H]⁺:477.19, found 477.19.

Example 161. Synthesis of Compound 505

To the solution of compound 504 (31.2 mg, 0.04 mmol) in DMA (2 mL) wasadded pentafluorophenyl ester 41a (27 mg, 0.04 mmol), followed by DIPEA(16 μL, 0.08 mmol). The reaction was stirred overnight and concentrated,purified by prep-HPLC with a gradient of MeCN/H₂O to give the titleproduct 505 (11.9 mg, 24%). ESI: m/z: calcd for C₆₃H₉₂N₁₁O₁₆S [M+H]⁺:1290.64, found 1290.64.

Example 162. Synthesis of Compound 508

To a solution of compound 300 (5.00 g, 12.1 mmol) in 10 mL DCM was added5 mL of TFA. The reaction mixture was stirred at r.t. for 1 h, and thenconcentrated. The crude product was dissolved in DCM (50 mL), to whichNHS (4.25 g, 37 mmol) and EDCI (7.10 g, 37 mmol) were added. Thereaction mixture was stirred at r.t. overnight, then concentrated andpurified by SiO₂ column with a gradient of DCM/MeOH to give the titlecompound 508 (5.00 g, 91%). ESI m/z calcd for C₂₅H₃₁N₂O₆ [M+H]⁺: 455.21,found 455.21.

Example 163. Synthesis of Compound 509

To a solution of compound 110 (1.00 g, 2.5 mmol, 1.0 eq.) in EtOH (10mL) were added compound 508 (1.80 g, 3.9 mmol, 1.5 eq.) and 0.1 MNaH₂PO₄ (2 mL) at r.t. The reaction mixture was stirred at r.t.overnight, and then concentrated. The residue was diluted with H₂O (100mL), then extracted with EtOAc (3×50 mL). The combined the organiclayers were dried over Na₂SO₄, filtered and concentrated, purified bySiO₂ column with a gradient of DCM/MeOH to give the title compound 509(0.93 g, 50%). ESI m/z calcd for C₄₂H₆₀N₃O₈ [M+H]⁺: 734.43, found734.43.

Example 164. Synthesis of Compound 510

In a hydrogenation bottle, Pd/C (0.093 g, 10 wt %) was added to asolution of compound 509 (0.93 g, 1.27 mmol) in EtOAc (20 mL). Themixture was shaken overnight under 1 atm H₂ then filtered through Celite(filter aid), the filtrate was concentrated to afford compound 510 (0.57g, 81%) and used in the next step without further purification. ESI m/zcalcd for C₂₈H₄₈N₃O₈ [M+H]⁺:554.34, found 554.34.

Example 165. Synthesis of Compound 511

To a solution of compound 510 (0.25 g, 0.45 mmol, 1.0 eq.) in EtOH (5mL) was added compound 502 (0.39 g, 0.68 mmol, 1.5 eq.) at r.t. Then0.1M NaH₂PO₄ (1 mL) was added, and the reaction mixture was stirred atr.t. overnight. The reaction mixture was concentrated under vacuum, andthe residue was diluted with H₂O (100 mL), then extracted with EtOAc(2×50 mL).

The combined organic layers were dried over Na₂SO₄, filtered andconcentrated, purified by SiO₂ column with a gradient of DCM/MeOH togive the title compound 511 (0.076 g, 17%). ESI m/z calcd forC₅₀H₇₄N₇O₁₅ [M+H]⁺: 1012.52, found 1012.53.

Example 166. Synthesis of Compound 512

To a solution of compound 511 (0.076 g, 75 mmol) in 2 mL DCM was added 4mL of TFA. The reaction mixture was stirred at r.t. for 1 h,concentrated, and the crude product 512 was used in the next stepwithout further purification. ESI m/z calcd for C₄₁H₅₈N₇O₁₃ [M+H]⁺:856.40, found 856.40.

Example 167. Synthesis of Compound 513

To a solution of above compound 512 in DMA (2 mL) were added compound41a (33 mg, 0.048 mmol) and DIPEA (25 uL, 0.144 mmol). The reaction wasstirred at r.t. for 3 h, then concentrated and purified by prep-HPLCwith a gradient of MeCN/H₂O to give the title compound 513 (21.3 mg,32%). ESI m/z calcd for C₆₆H₉₈N₁₁O₁₈S [M+H]⁺: 1364.67, found 1364.67.

Example 168. Synthesis of Compound 515

To a solution of compound 437 (1.00 g, 1.63 mmol) in 1 mL DCM was added2 mL TFA, the reaction mixture was stirred at r.t. for 1 h, and thenconcentrated. The resulting crude product 515 was used in the next stepwithout further purification. ESI m/z calcd for C₂₄H₃₂N₃O₆[M+H]⁺:458.22, found 458.22.

Example 169. Synthesis of Compound 516

To a solution of compound 515 in DMF (3 mL) were added pentafluorophenylester 41a (0.63 g, 0.91 mmol) and DIPEA (0.46 mL, 2.73 mmol). Thereaction was stirred at r.t. overnight, then concentrated and purifiedby SiO₂ column with a gradient of DCM/MeOH to give the title compound516 (1.75 g, theoretical yield) as a yellow oil. ESI m/z calcd forC₄₉H₇₂N₇O₁₁S [M+H]⁺:966.49, found 966.49.

Example 170. Synthesis of Compound 517

In a hydrogenation bottle, Pd/C (0.02 g, 10 wt %) was added to asolution of compound 516 (0.20 g, 0.20 mmol) in MeOH (15 mL). 1N HCl wasthen added to adjust pH to around 4. The mixture was shaken overnightunder 1 atm H₂ then filtered through Celite (filter aid), the filtratewas concentrated to afford compound 517, which was used in the next stepwithout further purification. ESI m/z calcd for C₄₁H₆₆N₇O₉S[M+H]⁺:832.46, found 832.46.

Example 171. Synthesis of Compound 519

To a solution of H-Dap(Boc)—OH(1.00 g, 4.9 mmol) in saturated NaHCO₃(20mL) at 0° C. was added compound 409 (2.30 g, 14.7 mmol). The reactionwas stirred at 0° C. for 1 h, then warmed to r.t. and stirred foranother hour. Then 1N KHSO₄ was added to adjust pH to −6 and theresulting mixture was extracted with EtOAc (2×50 mL). Combined organiclayers were dried over Na₂SO₄, filtered, and concentrated to givecompound 519 (0.42 g, 30% yield). ESI m/z calcd for C₁₂H₁₅N₂O₆ [M−H]⁻:283.10, found 283.10.

Example 172. Synthesis of Compound 520

To a solution of carboxylic acid 519 (0.21 g, 0.74 mmol) in EtOAc (10mL) were added pentafluorophenol (0.27 g, 1.48 mmol) and DCC (0.30 g,1.48 mmol). The reaction mixture was stirred at r.t. overnight and thenfiltered, with washing of the filter cake with EtOAc. The filtrate wasconcentrated to give the PFP-ester (0.17 g, 0.37 mmol), which wasdissolved in 1 mL DMF.

Compound 517 (0.36 g, 0.43 mmol) and DIPEA (0.13 mL, 0.74 mmol) wereadded and the reaction mixture was stirred at r.t. for 2 h. The reactionwas concentrated and purified by prep-HPLC with a gradient of MeCN/H₂Oto give the title compound 520 (50 mg, 13%). ESI m/z calcd forC₅₃H₈₀N₉O₁₄S [M+H]⁺: 1098.55, found 1098.55.

Example 173. Synthesis of Compound 521

To a solution of compound 520 (50 mg, 0.046 mmol) in 0.5 mL DCM wasadded 1 mL TFA. The reaction mixture was stirred at r.t. for 1 h, thenconcentrated, and purified by prep-HPLC with a gradient of MeCN/H₂O togive the title compound 521 (11 mg, 25%). ESI m/z calcd for C₄₈H₇₂N₉O₁₂S[M+H]⁺: 998.49, found 998.49.

Example 174. Synthesis of Compound 523

To a solution of compound 509 (1.00 g, 1.36 mmol) in 2 mL DCM was added4 mL TFA and the reaction mixture was stirred at r.t. for 1h, thenconcentrated to give compound 523, which was used in the next stepwithout further purification. ESI m/z calcd for C₃₃H₄₄N₃O₆[M+H]⁺:578.32, found 578.32.

Example 175. Synthesis of Compound 524

To the solution of compound 523 in DMF (5 mL) were addedpentafluorophenyl ester 41a (0.78 g, 1.13 mmol) and DIPEA (0.8 mL, 4.52mmol). The reaction was stirred at r.t. overnight and then concentrated,purified by SiO₂ column with a gradient of DCM/MeOH to give the titlecompound 524(1.64 g, theoretical yield). ESI m/z calcd for C₅₈H₈₄N₇O₁₁S[M+H]⁺:1086.59, found 1086.58.

Example 176. Synthesis of Compound 525

In a hydrogenation bottle, Pd/C (0.08 g, 10 wt %, 65.9% wet) was addedto a solution of compound 524 (0.80 g, 0.20 mmol) in MeOH (10 mL), and1N HCl was added to adjust pH to −4.

The mixture was shaken overnight under 1 atm H₂ then filtered throughCelite (filter aid), the filtrate was concentrated to afford compound525, which contained some un-reacted starting material and was used inthe next step without further purification. ESI: m/z: calcd forC₄₁H₆₆N₇O₉S [M+H]⁺:832.46, found 832.46.

Example 177. Synthesis of Compound 527

To a solution of H-Lys(Boc)—OH (1.00 g, 3.8 mmol, 1.0 eq.) in EtOH (16mL) was added compound 125 (1.00 g, 5.6 mmol, 1.5 eq.) at r.t. After 0.1M NaH₂PO₄ (3 mL) was added, the reaction mixture was stirred at r.t.overnight. The reaction was concentrated under vacuum, and the residueswas purified by SiO₂ column with a gradient of DCM/MeOH to give thetitle compound 527 (1.62 g, theoretical yield). ESI m/z calcd forC₁₉H₃₀N₃O₇ [M+H]⁺: 412.20, found 412.20.

Example 178. Synthesis of Compound 528

To a solution of carboxylic acid 527 (0.24 g, 0.58 mmol) in EtOAc (10mL) were added pentafluorophenol (0.21 g, 1.17 mmol) and DCC (0.24 g,1.17 mmol). The reaction mixture was stirred at r.t. overnight, and thenfiltered with washing of the filter cake with EtOAc, and the filtratewas concentrated. The resulting PFP-ester(32 mg, 0.056 mmol) wasdissolved in 1 mL DMF, to which compound 525 (50 mg, 0.056 mmol) andi-Pr₂EtN (29 μL, 0.168 mmol) were added. The reaction mixture wasstirred at r.t. for 2 h and concentrated. The residue was purified byHPLC with a gradient of MeCN/H₂O to give the title compound 528 (3 mg,4% yield). ESI m/z calcd for C₆₃H₉₉N₁₀O₁₇S [M+H]⁺: 1299.68, found1299.68.

Example 179. Synthesis of Compound 529

To a solution of compound 528 (3 mg, 0.002 mmol) in 0.5 mL DCM was added1 mL TFA, the reaction mixture was stirred at r.t. for 1h, thenconcentrated. The crude product was purified by HPLC with a gradient ofMeCN/H₂O to give the title compound 529 (1.43 mg, 52% yield). ESI m/zcalcd for C₅₈H₉₁N₁₀O₁₅S [M+H]⁺:1199.63, found 1199.62.

Example 180. Synthesis of Compound 532

The pentafluorophenyl ester of compound 527 (0.11 g, 0.19 mmol) wasdissolved in 1 mL DMF, to which compound 517 (0.21 g, 0.25 mmol) andi-Pr₂EtN (86 uL, 0.5 mmol) were added. The reaction mixture was stirredat r.t. for 2 h and concentrated. The residue was purified by prep-HPLCwith a gradient of MeCN/H₂O to give the title product 532 (20 mg, 9%).ESI m/z calcd for C₆₀H₉₃N₁₀O₅S [M+H]⁺: 1225.65, found 1225.66.

Example 181. Synthesis of Compound 533

To a solution of compound 532 (20 mg, 0.016 mmol) in 1 mL DCM was added2 mL TFA. The reaction mixture was stirred at rt for 1 h, thenconcentrated, and the crude product was purified by prep-HPLC with agradient of MeCN/H₂O to give the title compound 533 (8.9 mg, 18% yield).ESI m/z calcd for C₅₅H₈₅N₁₀O₁₃S [M+H]⁺:1125.59, found 1125.59.

Example 182. Synthesis of Compound 536

To a solution of H-Dap(Boc)—OH (1.00 g, 4.9 mmol, 1.0 eq.) in EtOH (30mL) was added compound 125 (2.00 g, 7.3 mmol, 1.5 eq.) at r.t. Then 0.1MNaH₂PO₄ (6 mL) was added, and the reaction mixture was stirred at r.t.overnight. The solvents were removed under vacuum, and the residues waspurified by SiO₂ column with a gradient of DCM/MeOH to give the titlecompound 536 (1.41 g, 78%). ESI m/z calcd for C₁₆H₂₄N₃O₇ [M+H]⁺: 370.15,found 370.15.

Example 183. Synthesis of Compound 537

To a solution of compound 536 (1.41 g, 3.8 mmol) in 2 mL DCM was added 5mL TFA. The reaction mixture was stirred at r.t. for 1h, and thenconcentrated. The crude product 537 was used in the next step withoutfurther purification. ESI m/z calcd for C₁₁H₁₆N₃O₅ [M+H]⁺:270.10, found270.10.

Example 184. Synthesis of Compound 538

To a solution of above compound 537 in EtOH (20 mL) was added compound125 (1.90 g, 6.9 mmol, 1.5 eq.) at r.t. Then 0.1M NaH₂PO₄ (4 mL) wasadded, and the reaction mixture was stirred at r.t. overnight. After thesolvents were removed under vacuum, then the residues was purified byHPLC with a gradient of H₂O/MeCN to give the title compound 538 (0.45 g,22% yield). ESI m/z calcd for C₁₉H₂₃N₄O₈ [M+H]⁺: 435.14, found 435.14.

Example 185. Synthesis of Compound 539

To a solution of compound 538 (0.15 g, 0.34 mmol), compound 438 (0.17 g,0.34 mmol) and HATU (0.16 g, 0.41 mmol) in DMF (2 mL), TEA (95 μL, 0.68mmol) was added. After stirring at r.t. for 1 h, the reaction wasconcentrated under reduced pressure and the residue was purified byprep-HPLC with a gradient of MeCN/H₂O to give the title compound 539 (34mg, 11% yield). ESI m/z calcd for C₄₄H₆₂N₇O₁₃ [M+H]⁺:896.43, found896.42.

Example 186. Synthesis of Compound 540

To a solution of compound 539 (34 mg, 0.04 mmol) in 0.5 mL DCM was added1 mL TFA. The reaction mixture was stirred at r.t. for 2h, and thenconcentrated to afford the title compound 540, which was used in thenext step without further purification. ESI m/z calcd for C₃₅H₄₆N₇O₁₁[M+H]⁺:740.30, found 740.32.

Example 187. Synthesis of Compound 541

To the solution of compound 540 in DMA (2 mL) was addedpentafluorophenyl ester 41a (28 mg, 0.04 mmol), followed by DIPEA (21μL, 0.12 mmol). The reaction was stirred overnight and then concentratedand purified by prep-HPLC with a gradient of MeCN/H₂O to give the titlecompound 541 (14.4 mg, 29%). ESI m/z calcd for C₆₀H₈₆N₁₁O₁₆S [M+H]⁺:1248.59, found 1248.60.

Example 188. Synthesis of Compound 544

To a solution of compound 132 (0.300 g, 0.329 mmol, 1.0 eq.) andtert-butyl (2-aminoethyl)carbamate hydrochloride (0.063 g, 0.395 mmol,1.2 eq.) in anhydrous DCM (30 mL) at 0° C. was added EDCI (0.189 g,0.988 mmol, 3.0 eq.). After stirring for 10 minutes, the reaction waswarmed to room temperature and stirred overnight. The reaction wasdiluted with DCM and washed with water and brine, dried over anhydrousNa₂SO₄, concentrated and purified by SiO₂ column chromatography(DCM/MeOH) to give compound 544 as a yellow foamy solid (0.132 g, 54%yield). ESI m/z calcd for C₅₂H₈₀N₉O₁₂S[M+H]⁺: 1054.6, found:1054.6.

Example 189. Synthesis of Compound 545

To a solution of compound 544 (0.132 g, 0.125 mmol, 1.0 eq.) in DCM (4.5mL) at r.t. was added TFA (1.5 mL) and stirred for 1 h. The reaction wasdiluted with anhydrous toluene and concentrated, and this operation wasrepeated for three times to give a yellow oil which was purified onprep-HPLC (C₁₈ column, mobile phase A: water, mobile phase B:acetonitrile, from 10% of B to 80% of B in 60 min). The fractions werepooled and lyophilized to give compound 545 (111 mg, 93% yield). ESI m/zcalcd for C₄₇H₇₂N₉O₁₀S [M+H]⁺: 954.5, found: 954.5.

Example 190. Synthesis of Compound 548

To a solution of compound 132 (0.050 g, 0.0549 mmol, 1.0 eq.) andtert-butyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate (0.024g, 0.0824 mmol, 1.5 eq.) in anhydrous DCM (10 mL) at 0° C. was addedEDCI (0.032 g, 0.1647 mmol, 3.0 eq.). After stirring for 10 minutes, thereaction was warmed to r.t. and stirred overnight. The mixture was thendiluted with DCM and washed with water and brine, dried over anhydrousNa₂SO₄, concentrated and purified by SiO₂ column chromatography(DCM/MeOH) to give the title compound as a yellow foamy solid (0.030 g,46% yield). ESI m/z calcd for C₅₈H₉₂N₉O₁₅S [M+H]⁺: 1186.6, found:1186.6.

Example 191. Synthesis of Compound 549

To a solution of compound 548 (0.030 g, 0.0253 mmol, 1.0 eq.) in DCM(3.0 mL) at r.t. was added TFA (1.0 mL). The reaction was stirred for 1h and then diluted with anhydrous toluene and concentrated, thisoperation was repeated for three times to give a yellow oil, which waspurified on prep-HPLC (C₁₈ column, mobile phase A: water, mobile phaseB: acetonitrile, from 10% of B to 80% of B in 60 min). The fractionswere pooled and lyophilized to give compound 549 (11.7 mg, 43% yield).ESI m/z calcd for C₅₃H₈₄N₉O₁₃S [M+H]⁺: 1086.6, found: 1086.6.

Example 192. Synthesis of Compound 552

To a solution of N-(2-aminoethyl)ethane-1,2-diamine (28.7 g, 275 mmol,10.0 eq.) and DMAP (0.034 g, 0.000275 mmol, 0.01 eq.) in anhydrous DCM(350 mL) at 0° C. was added Boc₂O (6.0 g, 0.0275 mmol, 1.0 eq.) inanhydrous DCM (100 mL) over 3 h. The reaction was then warmed to r.t.and stirred overnight, concentrated and purified by SiO₂ columnchromatography (DCM/MeOH) to give the title compound as a yellow oil(4.5 g, 80% yield). ESI m/z calcd for C₉H₂₂N₃O₂ [M+H]⁺: 204.2,found:204.2.

Example 193. Synthesis of Compound 553

To a solution of compound 132 (0.060 g, 0.0658 mmol, 1.0 eq.) andtert-butyl (2-((2-aminoethyl)amino)ethyl)carbamate (0.016 g, 0.0790mmol, 1.2 eq.) in anhydrous DCM (6 mL) at 0° C. was added EDCI (0.038 g,0.1974 mmol, 3.0 eq.). After stirring for 10 minutes, the reaction waswarmed to r.t. and stirred overnight. The mixture was concentrated andpurified on prep-HPLC (C₁₈ column, mobile phase A: water, mobile phaseB: acetonitrile, from 10% of B to 80% of B in 60 min). The fractionswere pooled and lyophilized to give the title compound 553 (48 mg, 66%yield). ESI m/z calcd for C₅₄H₈₅N₁₀O₁₂S [M+H]⁺: 1097.6, found: 1097.6.

Example 194. Synthesis of Compound 554

To a solution of compound 553 (0.048 g, 0.0437 mmol, 1.0 eq.) in DCM(3.0 mL) at r.t. was added TFA (1.0 mL). After stirring for 1 h, thereaction was diluted with anhydrous toluene and concentrated, and thisoperation was repeated for three times to give a yellow oil, which waspurified on prep-HPLC (C₁₈ column, mobile phase A: water, mobile phaseB: acetonitrile, from 10% of B to 80% of B in 60 min). The fractionswere pooled and lyophilized to give the title compound 554 (111 mg, 93%yield). ESI m/z calcd for C₄₉H₇₇N₁₀O₁₀S[M+H]⁺: 997.5, found: 997.5.

Example 195. Synthesis of Compound 558

To a solution of compound 132 (0.400 g, 0.439 mmol, 1.0 eq.) andH-Lys(Boc)—O′Bu·HCl (0.135 g, 0.528 mmol, 1.2 eq.) in anhydrous DCM (40mL) at 0° C. was added EDCI (0.189 g, 1.317 mmol, 3.0 eq.). Afterstirring for 10 min, the reaction was warmed to r.t. and stirredovernight. The mixture was diluted with DCM and washed with water andbrine, dried over anhydrous Na₂SO₄, concentrated and purified by SiO₂column chromatography (DCM/MeOH) to give compound 558 as a yellow oil(0.43 g, 82% yield). ESI m/z calcd for C₆₀H₉₄N₉O₁₄S [M+H]⁺: 1196.7,found:1196.7.

Example 196. Synthesis of Compound 559

To a solution of compound 558 (0.230 g, 0.192 mmol, 1.0 eq.) in DCM (6.0mL) at r.t. was added TFA (2.0 mL) and the reaction was stirred for 3 hand then diluted with toluene and concentrated, this operation wasrepeated for three times to give a yellow oil, which was purified onprep-HPLC (C₁₈column, mobile phase A: water, mobile phase B:acetonitrile, from 10% of B to 80% of B in 60 min). The fractions werepooled and lyophilized to give the title compound (153 mg, 76% yield).ESI m/z calcd for C₅₁H₇₈N₉O₁₂S [M+H]⁺: 1040.5, found:1040.5.

Example 197. Synthesis of Compound 562

To a solution of compound 558 (0.200 g, 0.167 mmol, 1.0 eq.) andBoc-L-Lys(Boc)—OH (0.070 g, 0.200 mmol, 1.2 eq.) in anhydrous DCM (10mL) at 0° C. was added HATU (0.095 g, 0.250 mmol, 1.5 eq.) and TEA (46μL, 0.334 mmol, 2.0 eq.). The reaction was stirred for 10 min at 0° C.and stirred for 10 minutes, then warmed to r.t. and stirred overnight.The mixture was diluted with DCM and washed with water and brine, driedover anhydrous Na₂SO₄, concentrated and purified by SiO₂ columnchromatography (DCM/MeOH) to give compound 562 as a colorless oil (0.270g, theoretical yield). ESI m/z calcd for C₇₆H₁₂₂N₁₁O₁₉S [M+H]⁺: 1524.9,found:1524.9.

Example 198. Synthesis of Compound 563

To a solution of compound 562 (0.270 g, 0.177 mmol, 1.0 eq.) in DCM (6.0mL) at r.t. was added TFA (2.0 mL) and stirred for 4 h. The mixture wasdiluted with anhydrous toluene and concentrated, this operation wasrepeated for three times to give a yellow oil, which was purified onprep-HPLC (C₁₈ column, mobile phase A: water, mobile phase B:acetonitrile, from 10% of B to 80% of B in 60 min). The fractions werepooled and lyophilized to give the title compound (172 mg, 83% yield).ESI m/z calcd for C₅₇H₉₀N₁₁O₁₃S [M+H]⁺: 1168.6, found:1168.6.

Example 199. Synthesis of Compound 566

To a solution of ethane-1,2-diamine (30.0 g, 0.5 mol, 10.0 eq.) inanhydrous DCM (500 mL) at 0° C. was added CbzCl (8.53 g, 0.050 mol, 1.0eq.) in anhydrous DCM (250 mL) over 7 h. The reaction was then warmed tor.t. and stirred overnight. The mixture was washed with water and brine,dried over anhydrous Na₂SO₄, and concentrated to give benzyl(2-aminoethyl)carbamate as a white solid (7.0 g, 94% yield). ESI m/zcalcd for C₁₀H₁₄N₂O₂ [M+H]⁺: 195.1, found:195.2.

Example 200. Synthesis of Compound 567

To a solution of compound 566 (7.0 g, 35.8 mmol, 1.0 eq.) and 37% HCHO(aq) (14 mL, 0.1772 mmol, 5.0 eq.) in MeOH (120 mL) at 0° C. was addedNaBH₃CN (3.9 g, 0.0620 mol, 1.6 eq.), then HOAc (3 mL) was added toadjust pH ˜7.0. The mixture was warmed to r.t. and stirred overnight,then concentrated. The residue was dissolved in DCM (200 mL), and washedwith water and brine, dried over anhydrous Na₂SO₄, concentrated andpurified by SiO₂ column chromatography (DCM/MeOH) to give the titlecompound as a light yellow oil (6.4 g, 80% yield). ESI m/: calcd forC₁₂H₁₈N₂O₂ [M+H]⁺: 224.1, found:224.1.

Example 201. Synthesis of Compound 568

Compound 567 (3.0 g, 13.4 mmol, 1.0 eq.) and Pd/C (0.3 g, 10% Pd/C, 50%wet) were mixed with HCl (3 mL) and MeOH (100 mL) in a hydrogenationbottle and shaken at 100 psi H₂ atmosphere for 5 h. Then the mixture wasfiltered over Celite and the filtrate was concentrated to give the titlecompound as a yellow solid (2.1 g, 98% yield). ¹H NMR (400 MHz, D₂O) δ3.33 (d, J=4.6 Hz, 2H), 3.27 (s, 2H), 2.79 (s, 6H).

Example 202. Synthesis of Compound 569

To a solution of compound 103 (0.58 g, 1.58 mmol, 1.0 eq.) and compound568 (0.051 g, 3.15 mmol, 2.0 eq.) in anhydrous DMF (10 mL) at 0° C. wereadded HATU (0.090 g, 2.37 mmol, 1.5 eq.) and TEA(0.656 mL, 4.74 mmol,3.0 eq.). After stirring for 10 minutes, the reaction was warmed to r.t.and stirred for 90 minutes. The mixture was diluted with H₂O andextracted with EA (3×100 mL). The combined organic layers were washedwith water and brine, dried over anhydrous Na₂SO₄, concentrated to givethe title compound as a yellow foamy solid (0.67 g, 97% yield). ESI m/zcalcd for C₂₁H₃₅N₄O₆ [M+H]⁺: 439.2, found:439.2.

Example 203. Synthesis of Compound 570

Pd/C (0.2 g, 10% Pd/C, 50% wet) was added to a solution of compound 569(0.60 g, 13.7 mmol, 1.0 eq.) in EA (10 mL). The mixture was shaken at100 psi H₂ atmosphere for 4 h. Then the mixture was filtered over Celiteand the filtrate was concentrated to give the title compound as a greenoil (5.50 g, 98% yield). ESI m/z calcd for C₂₁H₃₇N₄O₆₄ [M+H]⁺: 409.3,found:409.3.

Example 204. Synthesis of Compound 571

To a solution of compound 570 (0.50 g, 1.22 mmol, 1.0 eq.) in 95% EtOH(10 mL) and 0.1M NaH₂PO₄ (2 mL) was added compound 125 (0.683 g, 2.44mmol, 2.0 eq.) and the reaction was stirred overnight and thenconcentrated and purified by SiO₂ column chromatography (DCM/MeOH) togive the title compound as a light yellow oil (0.624 g, 89% yield). ESIm/z calcd for C₂₉H₄₄N₅O₇ [M+H]⁺: 574.3, found:574.3.

Example 205. Synthesis of Compound 572

To a solution of compound 571 (0.20 g, 0.349 mmol, 1.0 eq) in DCM (6.0mL) at r.t. was added TFA (2.0 mL) and the reaction was stirred for 2 h,then diluted with anhydrous toluene and concentrated, this operation wasrepeated for three times to give the title compound as a yellow oil (165mg, theoretical yield). ESI m/z calcd for C₂₄H₃₆N₅O₅ [M+H]⁺: 474.3,found:474.3.

Example 206. Synthesis of Compound 573

To a solution of compound 572 (0.165 g, 0.349 mmol, 1.0 eq.) inanhydrous DMF (2 mL) at 0° C. was added compound 41a (0.290 g, 1.047mmol, 1.2 eq.) in anhydrous DMF (3 mL) and the reaction was stirred for10 minutes, then warmed to r.t. and stirred for 1 h. The reactionmixture was concentrated and purified on prep-HPLC (C₁₈ column, mobilephase A: water, mobile phase B: acetonitrile, from 10% of B to 80% of Bin 60 min). The fractions were pooled and lyophilized to give the titlecompound (58 mg, 17% yield) as a light yellow foamy solid. ESI m/z calcdfor C₄₉H₇₆N₉O₁₀S [M+H]⁺: 982.5, found: 982.5.

Example 207. Synthesis of Compound 576

To a solution of 2-bromo-2-methylpropanoic acid (3.00 g, 17.9 mmol) inTHF (30 mL) was added trimethylamine (1M solution in THF, 17.9 mL, 35.9mmol). The reaction mixture was stirred overnight at r.t. Theprecipitate was collected by filtration and washed with EA to givecompound 576 (4.00 g, theoretical yield) as a white solid. ESI m/z calcdfor C₇H₁₆NO₂ [M+H]⁺: 146, found 146.

Example 208. Synthesis of Compound 577

To a solution of compound 576 (1.55 g, 6.9 mmol) and PFP (2.50 g, 13.8mmol) in DCM (20 mL) was added DCC (2.80 g, 13.8 mmol). The reactionmixture was stirred at r.t. overnight. The reaction was filtered and thefiltrate was concentrated under vacuum to give compound 577 as acolorless oil, which was used directly in the next step. ESI m/: calcdfor C₁₃H₁₅F₅NO₂ [M+H]⁺: 312, found 312.

Example 209. Synthesis of Compound 578

To a solution of compound 17 (1.78 g, 3.4 mmol) and the compound 577(6.9 mmol) in DMF (20 mL) was added DIPEA (1.8 mL, 10.4 mmol) at 0° C.The reaction mixture was warmed to r.t. and stirred for 1 h, thenconcentrated under vacuum and purified by silica column (100:1 to 5:1DCM/MeOH) to give compound 578 (1.20 g, 54% yield) as a foamy solid. ESIm/z calcd for C₃₂H₆₁N₄O₅SSi [M+H]⁺: 642, found 642.

Example 210. Synthesis of Compound 579

Compound 578 (1.20 g, 1.86 mmol) was dissolved in AcOH/THF/H₂O (v/v/v3:1:1, 20 mL) and stirred overnight. The reaction was then concentratedunder vacuum, and used for the next step without further purification.ESI m/z calcd for C₂₆H₄₇N₄O₅S [M+H]⁺: 527, found 527.

Example 211. Synthesis of Compound 580

To a solution of compound 579 (1.86 mmol) in 1,4-dioxane (10 mL) wasadded 1N NaOH (9.3 mL). And the reaction mixture was stirred for 2 h andconcentrated under vacuum. The residue was diluted with water (10 mL)and 1N HCl was added to adjust pH to −4. The mixture was concentratedunder vacuum to give compound 580 as a white solid. ESI m/z calcd forC₂₄H₄₃N₄O₅S [M+H]⁺: 499, found 499.

Example 212. Synthesis of Compound 581

To a solution of compound 580 (1.86 mmol) in pyridine (10 mL) was addedacetic anhydride (884 μL, 9.36 mmol) at 0° C. Then the reaction mixturewas warmed to r.t. and stirred overnight. The reaction was concentratedunder vacuum and then diluted with H₂O (20 mL) and washed with EA (3×10mL). The aqueous layer was concentrated under vacuum to give compound581 as a yellow solid. ESI m/z calcd for C₂₆H₄₅N₄O₆S [M+H]⁺: 541, found541.

Example 213. Synthesis of Compound 582

To a solution of compound 581 (150 mg, 0.277 mmol) and pentafluorophenol(76.5 mg, 0.415 mmol) in DCM (2 mL) was added EDCI (63.7 mg, 0.33 mmol).The reaction mixture was stirred for 3 h and concentrated under vacuumto give compound 582 as a yellow oil. ESI m/z calcd for C₃₂H₄₄F₅N₄O₆S[M+H]⁺:707, found 707.

Example 214. Synthesis of Compound 583

To a solution of compound 127 (50 mg, 0.07 mmol) and compound 582 (0.14mmol) in DMF (2 mL) was added DIPEA (49 μL, 0.28 mmol) at 0° C. Then thereaction mixture was warmed to r.t. and stirred for 1 h, thenconcentrated under vacuum and purified by prep-HPLC (10-90% MeCN/H₂O) togive compound 583 (30 mg, 46% yield) as a white solid. ESI m/z calcd forC₄₆H₆₈N₇O₁₁S [M+H]⁺:926, found 926.

Example 215. Synthesis of Compound 586

A suspension of betaine (870 mg, 7.4 mmol) in thionyl chloride (10 mL)was heated to 70° C. and stirred for 2 h. The reaction was concentratedunder vacuum and co-evaporated with toluene (3×10 mL) to afford compound586 as a yellow solid, which was used in the next step without furtherpurification.

Example 216. Synthesis of Compound 587

To a suspension of compound 17 (1.90 g, 3.71 mmol) in DCM (20 mL) wasadded DIPEA (2.58 mL, 14.8 mmol). Then the solution was cooled to 0° C.and the above compound 586 in DCM (20 mL) was added. The reactionmixture was warmed to r.t. and stirred for 1 h, concentrated undervacuum and purified by silica column (100:1 to 5:1 DCM/MeOH) to givecompound 587 (2.3 g, theoretical yield) as a yellow solid. ESI m/z calcdfor C₃₀H₅₇N₄O₅SSi [M+H]⁺:613, found 613.

Example 217. Synthesis of Compound 588

Compound 587 (2.3 g, 3.7 mmol) was dissolved in AcOH/THF/H₂O (v/v/v3:1:1, 40 mL) and stirred overnight. The reaction was concentrated togive compound 588, which was used in next step without any purification.ESI m/z calcd for C₂₄H₄₃N₄O₅S [M+H]⁺: 499, found 499.

Example 218. Synthesis of Compound 589

To a solution of compound 588 (3.7 mmol) in 1,4-dioxane (20 mL) wasadded 1N NaOH (18.5 mL), and the reaction mixture was stirred at r.t.for 2 h and concentrated under vacuum. The residue was diluted with 10mL water and acidified to pH ˜4 with 1N HCl, then concentrated to givecompound 589 (1.00 g, 57% yield) as a white solid. ESI m/z calcd forC₂₂H₃₉N₄O₅S [M+H]⁺: 471, found 471.

Example 219. Synthesis of Compound 590

To a solution of compound 589 (1.00 g, 2.12 mmol) in pyridine (10 mL)was added acetic anhydride (1 mL, 10.6 mmol) at 0° C. Then the reactionmixture was warmed to r.t. and stirred overnight. The reaction wasconcentrated under vacuum then diluted with water (20 mL) and washedwith EA (3×10 mL). The aqueous phase was concentrated under vacuum togive compound 590 as a yellow solid. ESI m/z calcd for C₂₄H₄₁N₄O₆S[M+H]⁺: 513, found 513.

Example 220. Synthesis of Compound 591

To a solution of compound 590 (70 mg, 0.136 mmol) and pentafluorophenol(30 mg, 0.163 mmol) in DCM (2 mL) was added DCC (33.7 mg, 0.163 mmol).The reaction mixture was stirred for 3 h and concentrated under vacuumto give the compound 591 as a yellow oil. ESI m/z calcd forC₃₀H₄₀F₅N₄O₆S [M+H]⁺:679, found 679.

Example 221. Synthesis of Compound 592

To a solution of compound 591 (0.136 mmol) and compound 127 (0.11 g,0.273 mmol) in DMF (2 mL) was added DIPEA (71 μL, 0.408 mmol) at 0° C.The reaction mixture was warmed to r.t. and stirred for 1 h,concentrated under vacuum and purified by prep-HPLC to give compound 592(30.9 mg, 25% yield) as a yellow solid. ESI m/z calcd for C₄₄H₆₄N₇O₁₁S[M+H]⁺:899, found 899.

Example 222. Synthesis of Compound 604

(S)—2-((tert-butoxycarbonyl)(methyl)amino)-3-methylbutanoic acid (33 mg,0.14 mmol), DCC (32 mg, 0.154 mmol) and pentafluorophenol (39 mg, 0.21mmol) were dissolved in ethyl acetate (20 mL) and the reaction wasstirred at room temperature overnight. The reaction was thenconcentrated to dryness to give compound 602, which was dissolved in 2mL of DMA, and a solution of compound 603 (52 mg, 0.14 mmol) in 3 mL ofDMA and DIPEA (48.5 μL, 0.28 mmol) were added. The reaction was stirredat room temperature overnight and then concentrated. The residue wasdiluted with 1 mL of acetonitrile and purified by reverse phase HPLCwith a gradient of MeCN/H₂O to afford compound 604 (40.2 mg, 490% yield)o ESI: m/z: calcd for C₂₈H₄₉N₄O₇S [M+H]⁺: 585.32, found 585.32.

Example 223. Synthesis of Compound 605

To a solution of compound 604 (40 mg, 0.069 mmol) in pyridine (8 mL) at0° C. was added acetic anhydride (20.4 mg, 0.2 mmol), and the reactionwas warmed to room temperature and stirred overnight, then concentrated.The residue was purified by column chromatography (MeOH/DCM) to affordthe title compound 605 (48.1 mg, −100% yield). ESI: m/z: calcd forC₃₀H₅₁N₄O₈S [M+H]⁺: 627.33, found 627.33.

Example 224. Synthesis of Compound 608

Compound 605 (48.1 mg, 0.077 mmol), DCC (17.4 mg, 0.085 mmol) andpentafluorophenol (21.2 mg, 0.115 mmol) were dissolved in ethyl acetate(10 mL) and the reaction was stirred overnight at room temperature, thenconcentrated to dryness to give compound 606, which was dissolved in 4mL of DMA, and a solution of compound 607 (20.7 mg 0.1 mmol) in 3 mL ofDMA and DIPEA (26.8 μL, 0.154 mmol) were added. The reaction was stirredat room temperature overnight and then concentrated. The residue wasdiluted with 1 mL of acetonitrile and purified by reverse phase HPLCwith a gradient of MeCN/H₂O to afford compound 608(63 mg, ˜100% yield).ESI: m/z: calcd for C₄₂H₆₆N₅O₉S [M+H]⁺: 816.45, found 816.45.

Example 225. Synthesis of Compound 609

Compound 608 from previous step was dissolved in DCM (1 mL) and treatedwith TFA (1 mL) at r.t. for 2 h. The reaction was concentrated and theresidue was dissolved in EtOH (20 mL). Compound 125 (30.8 mg, 0.11 mmol)and 0.1 M NaH₂PO₄ (4 mL) were added and the resulting mixture wasstirred at r.t. overnight, then concentrated and the residue waspurified by column chromatography (MeOH/DCM) to afford the titlecompound 609 (28.5 mg, 420% yield). ESI m/z: calcd for C₄₅H₆₅N₆O₁₀S[M+H]⁺: 881.44, found 881.44.

Example 226. Synthesis of Compound 612

To a solution of compound 608 (63 mg, 0.077 mmol) in DCM (1 mL) wastreated with TFA (1 mL) at room temperature for 2 h, then concentratedand the residue was dissolved in DMA (4 mL). Compound 611 (65.8 mg, 0.11mmol) and DIPEA (27 μL, 0.154 mmol) were added and the reaction wasstirred at room temperature overnight, then concentrated and the residuewas purified by reverse phase HPLC with a gradient of MeCN/H₂O to affordcompound 612 (14 mg, 16% yield). ESI: m/z: calcd for C₅₅H₈₄N₇O₁₆S[M+H]⁺: 1130.56, found 1130.57.

Example 227. Synthesis of Compound 614

To a solution of compound 436 (3.0 g, 4.00 mmol) in DMF (50 mL) wereadded HATU (2.3 g, 6 mmol) and TEA (1.7 mL, 12 mmol). The reaction wasstirred at 0° C. for 20 min and allowed to warm to r.t. and stirred for3h. After that, a solution of (4-aminophenyl)methanol (0.99 g, 8 mmol)in DMF (10 mL) was added, and the reaction was stirred at r.t. for 1.5h, then poured into a separatory funnel containing 150 mL of water andextracted with 50 mL of EtOAc twice. The organic phases were collectedand dried over anhydrous Na₂SO₄, filtered and concentrated. The residuewas purified by column chromatography (MeOH/DCM) to afford the titlecompound 614 (3.9 g, ˜100% yield). ESI: m/z: calcd for C₄₂H₆₈N₃O₁₅[M+H]⁺: 854.46, found 854.46.

Example 228. Synthesis of Compound 615

To a solution of compound 614 (1.9 g, 2.22 mmol) in MeOH (20 mL) wasadded Pd/C (0.19 g, 10 wt %) in a hydrogenation bottle. The mixture wasshaken for 50 min, filtered through Celite (filter aid), and thefiltrate was concentrated then dissolved in EtOH (100 mL). Compound 125(0.61 g, 2.20 mmol) and 0.1 M NaH₂PO₄ (20 mL) were added and thereaction was stirred overnight at room temperature, then concentrated.The residue was diluted with MeOH and purified by reverse phase HPLCwith a gradient of MeCN/H₂O to afforded compound 615 (0.30 g, 19%yield). ESI: m/z: calcd for C₄₂H₆₉N₄O₁₆ [M+H]⁺: 885.46, found 885.44.

Example 229. Synthesis of Compound 616

To a solution of compound 615 (0.12 g, 0.14 mmol) in DMF (50 mL) wasadded SOCl₂ (11 μL, 0.154 mmol) at 0° C. The reaction was stirred for 1hour at 0° C. and then concentrated to dryness to give crude productcompound 616 (0.13 g, 0.14 mmol). ESI: m/z: calcd for C₄₂H₆₈ClN₄O₁₅[M+H]⁺: 903.43, found 903.44.

Example 230. Synthesis of Compound 618

To a solution of compound 616 (0.13 g, 0.14 mmol) and compound 617 (0.06g, 0.07 mmol) in DMF (10 mL) were added TBAI (16 mg, 0.042 mmol) andDIPEA (64 μL, 0.35 mmol). The reaction was stirred at r.t. for 1 h andthen concentrated. The residue was purified by reverse phase HPLC with agradient of MeCN/H₂O to afford compound 618 (10 mg, 8.9% yield 0. ESI:m/z: calcd for C₇₉H₁₂₅N₉O₂₃S [M+H]⁺: 1599.85, found 1599.82.

Example 231. Synthesis of Compound 620

A mixture of tert-butyl 4-aminobutanoate (1.03 g, 6.12 mmol) andcompound 436 (4.16 g, 5.56 mmol) in DMF (18 mL)was cooled to 0° C. andHATU (2.32 g, 6.12 mmol) and TEA (1.2 mL, 8.34 mmol) were added insequence. The reaction was stirred for 50 min, then diluted with water(300 mL), and extracted with EtOAc (3×250 mL). The EtOAc solution waswashed with brine, dried over anhydrous Na₂SO₄, filtered, concentratedand purified by SiO₂ column chromatography (32:1 DCM/MeOH) to givecompound 620 (5.98 g). MS ESI m/z calcd for C₄₃H₇₅N₃O₁₆ [M+H]⁺ 890.51,found 891.09.

Example 232. Synthesis of Compound 621

To a solution of compound 620 (1.0 g, 1.13 mmol) in MeOH (50 mL) wasadded Pd/C (10 wt %, 0.10 g) in a hydrogenation bottle. The mixture wasshaken for 2 h, filtered through Celite (filter aid), and the filtratewas concentrated to afford compound 621(1.0 g, 1.32 mmol, yield>100%).ESI: m/z: calcd for C₃₅H₇₀N₃O₁₄ [M+H]⁺: 756.48, found 756.47.

Example 233. Synthesis of Compound 622

To a solution of compound 621 (0.93 g, 1.23 mmol, 1.0 eq) and compound125 (0.95 g, 1.84 mmol, 1.5 eq) in 95% EtOH (50 mL) at room temperaturewas added NaH₂PO₄ solution (0.1M, pH 5.0, 10 mL). The mixture wasstirred overnight, then concentrated and diluted with water (50 mL) andextracted with DCM (80 mL×3), dried over anhydrous Na₂SO₄, filtered,concentrated and purified by SiO₂ column chromatography (DCM:MeOH=25:1)to give the title compound as a light yellow oil (0.90 g, 80%). ESI m/z:calcd for C₄₃H₇₇N₄O₁₇ [M+H]⁺: 921.5, found: 921.5.

Example 234. Synthesis of Compound 623

Compound 622 (0.90 g, 0.98 mmol) was dissolved in HCOOH (50 mL) andstirred at room temperature for 1 hour. The reaction mixture wasconcentrated and co-evaporated with toluene twice, and the residue wasplaced on a vacuum pump to give compound 623 (0.85 g, 0.98 mmol, crudeproduct). ESI: m/z: calcd for C₃₉H₆₉N₄O₁₇ [M+H]⁺: 865.46, found 865.44.

Example 235. Synthesis of Compound 624

To a solution of compound 392 (92.9 mg, 0.236 mmol) and compound 623(510 mg, 0.59 mmol) in DMF (3 mL), were added HATU (179 mg, 0.472 mmol)and triethylamine (82 μL, 0.59 mmol) at 0° C. The reaction mixture waswarmed to r.t. and stirred for 1 h, then diluted with dichloromethane(50 mL), washed with 1N HCl (5 mL), water (5 mL), dried over sodiumsulfate, filtered and concentrated under vacuum and purified by silicagel column chromatography to afford the title product (295 mg, 60%yield). ESI m/z calcd for C₉₉H₁₆₈N₁₁O₃₆ [M+H]⁺: 2087.16, found 2087.14.

Example 236. Synthesis of Compound 625

To a solution of compound 624 (100 mg, 0.047 mmol) in DCM (3 mL) wasadded trifluoroacetic acid (3 mL), and the reaction mixture was stirredat room temperature for 2 hours, and then concentrated. The residue wasdissolved in DMF (3 mL), to which compound 41a (49.7 mg, 0.071 mmol) andDIPEA (12 μL, 0.071 mmol) were added. The reaction mixture was stirredat r.t. for 1h, then concentrated under vacuum and purified by prep-HPLCto afford the title compound (57 mg, 50% yield). ESI m/z calcd forC₁₁₅H₁₉₂N₁₁O₃₉S [M+H]⁺: 2439.31, found 2439.30.

Example 237. Synthesis of Compound 627

To a solution of undecanedioic acid (1.73 g, 8 mmol) in DMF (30 mL) wereadded K₂CO₃(1.1 g, 8 mmol) and BnBr (1.36 g, 8 mmol). The mixture wasstirred at r.t. overnight, then concentrated and purified by columnchromatography (PE/EtOAc) to afford the title compound 627 (1.1 g, 45%yield). ESI m/z: calcd for C₁₈H₂₇O₄ [M+H]⁺: 307.18, found 307.15.

Example 238. Synthesis of Compound 628

To a solution of compound 300 (2.00 g, 4.84 mmol) in DCM (5 mL) wasadded HCO₂H (5 mL). The reaction was stirred at room temperatureovernight, then concentrated to dryness and co-evaporated twice withDCM, and the residue was placed on a pump to give compound 628 (1.72 g,˜100% yield). ESI m/z calcd for C₂₁H₂₇NO₄ [M+H]⁺: 358.19, found 358.19.

Example 239. Synthesis of Compound 629

To a solution of compound 301 (1.12 g, 4.83 mmol) and compound 628 (1.72g, 4.83 mmol) in DCM (30 mL) were added HATU (1.83 g, 4.83 mmol) and TEA(0.68 mL, 4.83 mmol) at 0° C. The reaction was warmed to r.t. andstirred for 1 h, then diluted with 50 mL DCM and poured into aseparatory funnel containing 50 mL of water. The organic phase wasseparated, and washed with brine (50 mL), dried over anhydrous Na₂SO₄,filtered and concentrated. The residue was purified by columnchromatography (MeOH/DCM) to afford the title compound 629 (2.21 g, 80%yield). ESI m/z calcd for C₃₂H₄₈N₂O₇ [M+H]⁺: 573.35, found 573.35.

Example 240. Synthesis of Compound 630

To a solution of compound 629 (2.21 g, 3.86 mmol) in MeOH (20 mL) wasadded Pd/C (10 wt %, 0.2 g) in a hydrogenation bottle. The mixture wasstirred under 1 atm H₂ overnight, filtered through Celite (filter aid),and the filtrate was concentrated to afford compound 630 (1.5 g, ˜100%yield). ESI m/z calcd for C₁₈H₃₆N₂O₇ [M+H]⁺: 393.25, found 393.25Example 241. Synthesis of compound 631

To a solution of compound 630 (1.50 g, 3.86 mmol) and compound 627 (1.10g, 3.6 mmol) in DCM (50 mL) were added HATU (1.48 g, 3.9 mmol) and TEA(0.55 mL, 3.9 mmol) at 0° C. The reaction mixture was stirred at r.t.for 1 h, then diluted with 50 mL DCM and poured into a separatory funnelcontaining 50 mL of water. The organic phase was separated, washed withbrine (50 mL), dried over anhydrous Na₂SO₄, filtered and concentrated.The residue was purified by column chromatography (MeOH/DCM) to affordthe title compound 631 (1.50 g, 61% yield). ESI m/z calcd forC₃₆H₆₁N₂O₁₀ [M+H]⁺: 681.42, found 681.42.

Example 242. Synthesis of Compound 632

To a solution of compound 631 (1.50 g, 2.2 mmol) in DCM (1 mL) was addedTFA (3 mL). The reaction was stirred at room temperature for 1 h, thenconcentrated to dryness and co-evaporated twice with DCM, and theresidue was placed on a pump to give compound 632 (0.09 g, 2.2 mmol,crude product). ESI m/z: calcd for C₃₂H₅₃N₂O₁₀ [M+H]⁺: 625.36, found625.35.

Example 243. Synthesis of Compound 633

To a solution of compound 632 (1.50 g, 2.20 mmol) and Z-Lys-OH (0.62 g,2.20 mmol) in DCM (50 mL) were added HATU (0.84 g, 2.20 mmol) and TEA(0.31 mL, 2.20 mmol) at 0° C. The reaction mixture was stirred at r.t.for 1 h, then diluted with 50 mL DCM and poured into a separatory funnelcontaining 100 mL of water. The organic phase was separated, and washedwith brine (100 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated. The residue was purified by column chromatography(MeOH/DCM) to afford the title compound 633 (1.00 g, 53% yield). ESI m/zcalcd for C₄₆H₇₁N₄O₁₃ [M+H]⁺: 887.49, found 887.50.

Example 244. Synthesis of Compound 634

To a solution of compound 633 (0.50 g, 0.56 mmol) in DMF (5 mL) wasadded HATU (0.21 g, 0.56 mmol) and the reaction was stirred at roomtemperature for 30 min. After that, a solution of compound 438 (0.27 g,0.56 mmol) in DMF (5 mL) and TEA (85 μL, 0.6 mmol) were added insequence at 0° C., and the reaction was stirred for 1 h. The reactionmixture was poured into a separatory funnel containing 100 mL of waterand extracted with 50 mL of EtOAc twice. The organic phase was washedonce with 100 mL of brine, dried over anhydrous Na₂SO₄, filtered andconcentrated. The residue was purified by column chromatography(MeOH/DCM) to afford the title compound 634 (0.40 g, 55% yield). ESIm/z: calcd for C₇₁H₁₁₀N₇O₁₈ [M+H]⁺: 1348.78, found 1348.78.

Example 245. Synthesis of Compound 635

To a solution of compound 634 (0.40 g, 0.30 mmol) in MeOH (20 mL) wasadded Pd/C (10 wt %, 0.2 g) in a hydrogenation bottle. The mixture wasstirred under 1 atm H₂ overnight, filtered through Celite (filter aid),and the filtrate was concentrated and re-dissolved in EtOH (20 mL).Compound 125 (88.5 mg, 0.30 mmol) and 0.1 M NaH₂PO₄ (4 mL) were added.The mixture was stirred at r.t. overnight, then concentrated and theresidue was purified by column chromatography (MeOH/DCM) to afford thetitle compound 635 (0.10 g, 26% yield). ESI m/z: calcd for C₆₄H₁₀₆N₉O₁₉[M+H]⁺: 1304.75, found 1304.75.

Example 246. Synthesis of Compound 636

To a solution of compound 635 (0.10 g, 0.077 mmol) in DCM (1 mL) wasadded TFA (3 mL). The reaction was stirred at room temperature for 30min, then concentrated to dryness and co-evaporated twice with DCM, andthe residue was dissolved in DMA (4 mL). Compound 41a (65.8 mg, 0.11mmol) and DIPEA (26 μL, 0.15 mmol) were added. The reaction mixture wasstirred at room temperature for 1 h and then concentrated. The residuewas diluted with 2 mL MeCN and purified by reverse phase HPLC with agradient of MeCN/H₂O to afforded compound 636 (20 mg, 15% yield). ESIm/z: calcd for C₈₀H₁₃₀N₁₃O₂₂S [M+H]⁺: 1656.90, found 1656.91.

Example 247. Synthesis of Compound 638

To a solution of compound 633 (0.50 g, 0.56 mmol) in DMF (5 mL) wasadded HATU (0.21 g, 0.56 mmol) and the reaction was stirred at roomtemperature for 30 min. After that, a solution of compound 110 (0.22 g,0.56 mmol) in DMF (5 mL) and TEA (85 μL, 0.60 mmol) were added at 0° C.After stirring for 1 h, the reaction mixture was poured into aseparatory funnel containing 100 mL of water and extracted with 50 mL ofEtOAc twice. The organic phase was separated and washed with 100 mL ofbrine, dried over anhydrous Na₂SO₄, filtered and concentrated. Theresidue was purified by column chromatography (MeOH/DCM) to afford thetitle compound 638 (0.20 g, 26% yield). ESI m/z: calcd for C₆₇H₁₀₃N₆O₁₇[M+H]⁺: 1263.73, found 1263.73.

Example 248. Synthesis of Compound 639

To a solution of compound 638 (0.20 g, 0.16 mmol) in MeOH (20 mL) wasadded Pd/C (10 wt %, 0.2 g) in a hydrogenation bottle. The mixture wasstirred under 1 atm H₂ overnight, filtered through Celite (filter aid),and the filtrate was concentrated then dissolved in EtOH (20 mL).Compound 125 (47.2 mg, 0.30 mmol) and 0.1 M NaH₂PO₄ (4 mL) were added.The mixture was stirred at r.t. overnight, then concentrated and theresidue was purified by column chromatography (MeOH/DCM) to afford thetitle compound 639 (75 mg, 40% yield). ESI m/z: calcd for C₆₀H₉₈N₇O₁₈[M+H]⁺: 1204.69, found 1204.68.

Example 249. Synthesis of Compound 640

To a solution of compound 639 (75 mg, 0.06 mmol) in DCM (1 mL) was addedTFA (3 mL). The reaction was stirred at room temperature for 30 min,then concentrated to dryness and co-evaporated twice with DCM, and theresidue was dissolved in DMA (2 mL). Compound 41a (41 mg, 0.06 mmol) andDIPEA (26 μL, 0.15 mmol) were added and the reaction mixture was stirredat room temperature for 1 h, then concentrated. The residue was dilutedwith 2 mL MeCN and purified by reverse phase HPLC with a gradient ofMeCN/H₂O to afford compound 640 (34 mg, 37% yield). ESI m/z: calcd forC₇₆H₁₂₂N₁₁O₂₁S [M+H]⁺: 1556.85, found 1556.85.

Example 250. Synthesis of Compound 642

To a solution of diethylene glycol (20 g, 0.188 mol) in THF (200 mL) wasadded Na (0.43 g, 0.018 mol). After stirring at r.t. for 1 h, tert-butylacrylate (48 g, 0.376 mol) was added and the reaction mixture wasstirred at r.t. for 2 days. The reaction was concentrated under vacuumand purified by column chromatography to afford the title compound (34g, 50% yield). ESI m/z calcd for C₁₈H₃₅O₇ [M+H]⁺: 363.23, found 363.23.

Example 251. Synthesis of Compound 643

Compound 642 (34 g, 0.093 mol) was dissolved in formic acid (100 mL) atroom temperature and stirred overnight. The reaction was concentratedunder vacuum to afford the title compound. ESI m/z calcd for C₁₀H₁₉O₇[M+H]⁺: 251.11, found 251.11.

Example 252. Synthesis of Compound 644

To a solution of amine 630 (1.50 g, 3.82 mmol) and diacid 643 (1.90 g,7.64 mmol) in DMF (10 mL) were added HATU (1.45 g, 3.82 mmol) and DIPEA(0.66 mL, 3.82 mmol) at 0° C. The reaction mixture was warmed to r.t.and stirred for 1 h, then diluted with DCM (80 mL), washed with water(10 mL), dried over sodium sulfate, filtered, concentrated and purifiedby silica gel column chromatography to afford a colorless liquid (1.75g, 75% yield). ESI m/z calcd for C₂₈H₅₃N₂O₁₃ [M+H]⁺: 625.35, found625.35.

Example 253. Synthesis of Compound 645

To a solution of compound 644 (1.75 g, 2.8 mmol) in DCM (20 mL) wereadded EDCI (1.07 g, 5.6 mmol) and NHS (0.64 g, 5.6 mmol) at 0° C. Thereaction was warmed to room temperature and stirred overnight, thendiluted with DCM (80 mL), washed with water (10 mL), dried over sodiumsulfate, filtered and concentrated under vacuum to afford the titlecompound (2.00 g, ˜100% yield). ESI m/z calcd for C₃₂H₅₆N₃O₁₅ [M+H]⁺:722.36, found 722.36.

Example 254. Synthesis of Compound 646

To a solution of N-α-Cbz-L-lysine (1.17 g, 4.2 mmol) in water (10 mL)was added sodium bicarbonate (0.47 g, 5.6 mmol), and the reactionmixture was cooled to 5° C., and compound 645 (2.00 g, 2.8 mmol)dissolved in 1,4-Dioxane (10 mL) was added. The reaction was warmed tor.t. and stirred for 1 h, then acidified to pH 3 by addition of 1 N HCl,extracted with DCM (50 mL×3). The organic extracts were washed withwater (20 mL), dried over sodium sulfate, filtered and concentrated toafford the title product (2.3 g, 92% yield). ESI m/z calcd forC₄₂H₇₁N₄O₁₆ [M+H]⁺: 887.48, found 887.48.

Example 255. Synthesis of Compound 647

To a solution of amine 438 (1.87 g, 3.9 mmol) and acid 646 (2.3 g, 2.59mmol) in dichloromethane (30 mL) were added HATU (0.98 g, 2.59 mmol) andDIPEA (450 μL, 2.59 mmol) at 0° C. The reaction mixture was warmed tor.t. and stirred for 1 h, then concentrated under vacuum and purified bysilica gel column chromatography to afford the title compound (2.4 g,70% yield). ESI m/z calcd for C₆₇H₁₁₀N₇O₂₁ [M+H]⁺: 1348.77, found1348.77.

Example 256. Synthesis of Compound 648

To a solution of compound 647 (2.4 g, 1.78 mmol) in MeOH (20 mL) wasadded Pd/C (10 wt %, 0.2 g) in a hydrogenation bottle. The mixture wasstirred under 1 atm H₂ overnight, filtered through Celite (filter aid),and the filtrate was concentrated and re-dissolved in EtOH (20 mL).Compound 125 (0.79 g, 2.67 mmol) and 0.1 M NaH₂PO₄ (10 mL) were addedand the mixture was stirred at r.t. overnight, then concentrated and theresidue was purified by column chromatography (MeOH/DCM) to afford thetitle compound 648 (1.52 g, 62% yield). ESI m/z: calcd for C₆₇H₁₁₁N₈O₂₂[M+H]⁺: 1379.77, found 1379.75.

Example 257. Synthesis of Compound 649

To a solution of the compound 648 (51 mg, 0.037 mmol) in dichloromethane(2 mL) was added trifluoroacetic acid (2 mL), and the reaction mixturewas stirred at room temperature for 2 hours, then concentrated andre-dissolved in DMF (1.0 mL). A solution of compound 41a (38.4 mg, 0.055mmol) in DMF (1.0 mL) was added at 0° C., followed by DIPEA (13 μL,0.074 mmol). The reaction mixture was then warmed to r.t. and stirredfor 1 h, concentrated under vacuum and purified by prep-HPLC to affordthe title compound 649 (36.5 mg, 60% yield). ESI m/z calcd forC₇₉H₁₂₇N₁₂O₂₅S [M+H]⁺: 1675.86, found 1675.86.

Example 258. Synthesis of Compound 651

To a solution of N-Phthaloylglycine (10.0 g, 48.7 mmol) in DCM (100 mL)was added oxalyl chloride (6.3 mL, 73.1 mmol) at r.t., followed by adrop of DMF. The reaction was stirred for 2 h and then concentrated togive compound 651(10.8 g) as a yellow solid.

Example 259. Synthesis of Compound 652

To a solution of Boc-hydrazine (7.08 g, 53.5 mmol) in DCM (200 mL) wasadded Et₃N (13.5 mL, 97.4 mmol), and then compound 651 (10.8 g, 48.7mmol) was added at 0° C. After that the reaction was stirred at r.t. for30 min. and poured into ice-water (100 mL) and extracted with DCM (3×100mL). The combined organic phases were washed with water (100 mL) andbrine (100 mL), dried over anhydrous Na₂S₄, filtered and concentrated togive a white solid (15.5 g, 100% yield). ESI m/z calcd for C₁₅H₁₈N₃O₅[M+H]⁺: 320.12, found: 320.12.

Example 260. Synthesis of Compound 653

Compound 652 (15.5 g, 48.7 mmol) was dissolved in DCM (150 mL) andtreated with TFA (50 mL) at r.t. for 1 h, then concentrated to give awhite solid (10.6 g, 100% yield). ESI m/z calcd for C₁₀H₁₀N₃O₃ [M+H]⁺:220.06, found: 220.06.

Example 261. Synthesis of Compound 654

To a solution of compound 653 (10.6 g, 48.7 mmol) in DCM (200 mL) wasadded Et₃N (13.5 mL, 97.4 mmol) and compound 651 (10.8 g, 48.7 mmol) at0° C. The reaction was warmed to r.t. and stirred overnight. Theprecipitate was collected by filtration and suspended in water (100 mL)and stirred for 20 min. The mixture was filtered again to give a whitesolid (15.7 g, 80% yield). ESI m/z calcd for C₂₀H₁₅N₄O₆ [M+H]⁺: 407.09,found: 407.09.

Example 262. Synthesis of Compound 655

NaH (0.5 g, 12.3 mmol) was added to a solution of compound 654 (2.0 g,4.92 mmol) in DMF (40 mL) in portions at 0° C. The mixture was warmed tor.t. and stirred for 3 h. After that tert-butyl bromoacetate (2.0 g,10.3 mmol) was added and the reaction was stirred overnight beforepouring into ice-water (100 mL) and extraction with DCM (3×50 mL). Thecombined organic phase was washed with water (50 mL), brine (50 mL),dried over anhydrous Na₂SO₄, filtered and concentrated, purified bysilica gel chromatography to give a white solid (1.5 g, 50% yield). ESIm/z calcd for C₃₂H₃₅N₄O₁₀ [M+H]⁺: 635.23, found: 635.23.

Example 263. Synthesis of Compound 656

A mixture of compound 655 (1.5 g, 2.36 mmol) and hydrazine (442 mg, 7.08mmol) in ethanol (30 mL) was refluxed for 1 h, then cooled to r.t. andfiltered. The filtrate was concentrated and taken up in ethyl acetate(20 mL), filtered again. The filtrate was concentrated to give a whitesolid (750 mg, 85% yield). ESI m/z calcd for C₁₆H₃₁N₄O₆ [M+H]⁺: 375.22,found: 375.22.

Example 264. Synthesis of Compound 657

A solution of compound 656 (750 mg, 2 mmol) in THF (2 mL) was added tosaturated NaHCO₃ aqueous solution (30 mL) and then cooled to 0° C.,compound 409 (622 mg, 4 mmol) was then added and the reaction wasstirred at 0° C. for 1 h. A white solid was collected by filtration (854mg, 80% yield). ESI m/z calcd for C₂₄H₃₁N₄O₁₀ [M+H]⁺: 535.20, found:535.20.

Example 265. Synthesis of Compound 658

Compound 657 (854 mg, 1.6 mmol) was dissolved in DCM (3 mL) and treatedwith TFA (3 mL) at r.t. for 2 h. The reaction was then concentrated togive compound 658 (675 mg, 100% yield). ESI m/z calcd for C₁₆H₁₅N₄O₁₀[M+H]⁺: 423.07, found: 423.07.

Example 266. Synthesis of Compound 659

To a solution of compound 658 (200 mg, 0.47 mmol) in DMF (5 mL) wasadded tert-butyl 4-Aminobutanoate (158 mg, 0.99 mmol) and EDC (189.7 mg,0.99 mmol) at 0° C. The reaction was warmed to r.t. and stirredovernight, poured into ice-water, and extraction with DCM (3×10 mL). Thecombined organic phase was washed with 1 N HCl (5 mL), water (5 mL),brine (5 mL), dried over anhydrous Na₂SO₄, filtered and concentrated togive a white solid (330 mg, 100% yield).

Example 267. Synthesis of Compound 660

Compound 659 (330 mg, 0.47 mmol) was dissolved in DCM (3 mL) and treatedwith TFA (3 mL) at r.t. for 2 h. The reaction was concentrated andre-dissolved in DMF (5 mL) and cooled to 0° C., NHS (113 mg, 0.98 mmol)and EDC (189 mg, 0.98 mmol) were added in sequence. The reaction waswarmed to r.t. and stirred overnight, poured into ice-water, andextraction with DCM (3×20 mL). The combined organic phase was washedwith water (5 mL), brine (5 mL), dried over anhydrous Na₂SO₄, filteredand concentrated to give a white solid (369 mg, 100% yield). ESI m/zcalcd for C₃₂H₃₅N₈O₁₆ [M+H]⁺: 787.21, found: 787.21.

Example 268. Synthesis of Compound 663

Compound 633 (200 mg, 0.225 mmol) was dissolved in DMF (5 mL) and cooledto 0° C., tert-butyl 4-Aminobutanoate (71.8 mg, 0.45 mmol) and EDC (86.2mg, 0.45 mmol) were added in sequence. The reaction was warmed to r.t.and stirred overnight, poured into ice-water, and extraction with DCM(3×10 mL). The combined organic phase was washed with water (5 mL),brine (5 mL), dried over anhydrous Na₂SO₄, filtered and concentrated togive compound 663 (231 mg, 100% yield). ESI m/z calcd for C₅₄H₈₆N₅O₁₄[M+H]⁺:1028.61, found:

Example 269. Synthesis of Compound 664

Compound 663 (231 mg, 0.225 mmol) was dissolved in DCM (3 mL) andtreated with TFA (3 mL) at r.t. for 1 h. The reaction was concentratedand re-dissolved in DMF (5 mL) and cooled to 0° C., compound 110 (44 mg,0.112 mmol), HATU (85.5 mg, 0.225 mmol) and DIPEA (39 μL, 0.225 mmol)were added in sequence. The reaction was warmed to r.t. and stirredovernight, poured into ice-water, and extraction with DCM (3×10 mL). Thecombined organic phase was washed with 1 N HCl (5 mL), water (5 mL),brine (5 mL), dried over anhydrous Na₂SO₄, filtered and concentrated,purified by silica gel column chromatography (0-5% MeOH/DCM) to give awhite foam (206 mg, 80% yield). ESI m/z calcd for C₁₂₁H₁₈₅N₁₂O₃₁ [M+H]⁺:2302.32, found: 2302.34.

Example 270. Synthesis of Compound 665

Compound 664 (206 mg, 0.089 mmol) was dissolved in MeOH (5 mL) and mixedPd/C (10 wt %, 20 mg), hydrogenated under 1 atm H₂ pressure overnight.The mixture was then filtered through Celite (filter aid), and thefiltrate was concentrated to afford compound 665 (165 mg, 100% yield).ESI m/z calcd for C₉₁H₁₆₁N₁₂O₂₇ [M+H]⁺: 1854.15, found 1854.15.

Example 271. Synthesis of Compound 666

To a solution of compound 665 (165 mg, 0.089 mmol) in ethanol (10 mL)were added compound 660 (140 mg, 0.178 mmol) and phosphate buffer (0.5M,pH 7.5, 3 mL) at 0° C. The reaction was stirred at r.t. overnight andthen concentrated and purified by silica gel column chromatography (0-5%MeOH/DCM) to give compound 666 (128 mg, 61% yield). ESI m/z calcd forC₁₁₅H₁₈₅N₁₈O₃₇ [M+H]⁺: 2410.31, found: 2410.31.

Example 272. Synthesis of Compound 667

Compound 666 (128 mg, 0.053 mmol) was dissolved in DCM (3 mL) andtreated with TFA (3 mL) at r.t. for 2 h. The reaction was concentratedand co-evaporated with DCM for three times to give compound 667 (120 mg,100% yield). ESI m/z calcd for C₁₀₆H₁₆₉N₁₈O₃₅ [M+H]⁺: 2254.19, found:2254.19.

Example 273. Synthesis of Compound 668

Compound 667 (120 mg, 0.053 mmol) and compound 41a (36.6 mg, 0.053 mmol)were dissolved in DMA (5 mL) and cooled to 0° C. DIPEA (18 μL, 0.106mmol) was added and the reaction was warmed to r.t. and stirred for 1 h.After the reaction mixture was concentrated, the residue was purified byprep-HPLC (C₁₈, 10-90% acetonitrile/water) to give compound 668 (73 mg,50% yield). ESI m/z calcd for C₁₃₁H₂₀₉N₂₂O₄₀S [M+H]⁺: 2762.46, found:2762.46.

Example 274. Synthesis of Compound 670

To the solution of compound 621 (1 g, 1.32 mmol) in a saturated solutionof NaHCO₃(20 mL) was added compound 409 (0.4 g, 2.64 mmol) in ice-waterbath. The reaction was stirred for 30 min and then poured into aseparatory funnel containing 100 mL of ethyl acetate and the organicphase was separated, washed with 50 mL of water and 50 mL of brine,dried over anhydrous Na₂SO₄, filtered and concentrated to give compound670 (0.8 g, yield 72%). ESI: m/z: caled for C₃₉H₇₀N₃O₁₆ [M+H]⁺: 836.47,found 836.47.

Example 275. Synthesis of Compound 671

Compound 670 (0.9 g, 0.98 mmol) was dissolved in HCOOH (50 mL) andstirred at room temperature for 1 hour. The reaction mixture wasconcentrated and co-evaporated with toluene twice, and the residue wasplaced on a vacuum pump to give an oil. Half of the material wasdissolved in DMF (10 mL) and compound 110 (0.35 g, 0.48 mmol), HATU(0.36 g, 0.96 mmol) and TEA (0.15 mL, 1.44 mmol) were added at 0° C.After stirring for 30 min, the reaction mixture was poured into aseparatory funnel containing 100 mL of water and extracted twice with 50mL of ethyl acetate. The organic phases were combined, dried overanhydrous Na₂SO₄, filtered and concentrated. The residue was purified bycolumn chromatography (MeOH/DCM) to afford the title compound 671 (21mg, 2%). ESI: m/z: calcd for C₉₁H₁₅₃N₈O₃₅ [M+H]⁺: 1919.04, found1919.04.

Example 276. Synthesis of Compound 672

To a solution of compound 671 (21 mg, 0.01 mmol) in DCM (0.5 mL) wasadded TFA (1 mL). The reaction was stirred at room temperature for 1hour, and then concentrated and co-evaporated twice with DCM, and theresidue was placed on a vacuum pump for 2 h, and then dissolved in DMA(2 mL). Compound 41a (6.9 mg, 0.01 mmol) was added, followed by DIPEA(17 μL, 0.1 mmol). The reaction mixture was stirred at room temperaturefor 30 min and then concentrated. The residue was purified by reversephase HPLC with a gradient of MeCN/H₂O to afforded compound 672 (10 mg,44% yield). ESI m/z: calcd for C₁₀₇H₁₇₇N₁₂O₃₈S [M+H]⁺:2271.20, found2271.20.

Example 277. Synthesis of Compound 675

A mixture of compound 621 (5.98 g, 6.73 mmol) and Pd/C (10 wt %, 0.6 g)in methanol (30 mL) was hydrogenated under 1 atm H₂ pressure overnightand then filtered through Celite (filter aid). The filtrate wasconcentrated and re-dissolved in TI-IF (60 mL), compound 674 (1.01 g,2.42 mmol) and HOBt (817 mg, 6.05 mmol) were added at 0° C. DCC (1.25 g,6.05 mmol) and DIPEA (2.1 mL, 12.10 mmol) were added in sequence. Thereaction was stirred at r.t. overnight, then diluted with EtOAc (400mL), and washed with 1N HCl, saturated sodium bicarbonate and brine,dried over anhydrous Na₂SO₄, filtered, concentrated and purified by SiO₂column chromatography (24:1 DCM/MeOH) to give compound 675 (5.65 g, 49%yield). MS ESI m/z calcd for C₉₀H₁₅₄N₈O₃₄ [M+H]⁺ 1892.06, found 1892.83.

Example 278. Synthesis of Compound 676

A mixture of compound 675 (3.71 g, 1.96 mmol) and Pd/C (10 wt %, 0.40 g)in methanol (50 mL) was hydrogenated under 1 atm H₂ pressure overnightand then filtered through Celite (filter aid). The filtrate wasconcentrated to afford compound 676 (4.57 g, 51% yield). MS ESI m/zcalcd for C₇₄H₁₄₂N₈O₃₀ [M+H]⁺ 1623.98, found 1624.42.

Example 279. Synthesis of Compound 677

To a solution of compound 676 (315 mg, 0.194 mmol) in EtOH (10 mL) wereadded Na₂HPO₄ aqueous solution (0.5 M, 2.5 mL) and compound 125 (136 mg,0.485 mmol). The mixture was stirred at room temperature for 3 days,concentrated and purified by SiO₂ column chromatography (3:2 H₂O/MeCN)to give an oil (50 mg, 13% yield), which was dissolved indichloromethane (5 mL) and treated with TFA (5 mL) at r.t. overnight,and then concentrated to afford compound 677 (47 mg, 98% yield). MS ESIm/z calcd for C₈₂H₁₄₀N₁₀O₃₆ [M+H]⁺ 1841.94, found 1841.88.

Example 280. Synthesis of Compound 678

A mixture of compound 677 (154 mg, 0.0837 mmol) and compound 110 (33 mg,0.0837 mmol) in DMF (6 mL) was cooled to 0° C. and HATU (64 mg, 0.167mmol,) and TEA (46 μL, 0.335 mmol) were added in sequence. The reactionwas stirred for 1 h then diluted with water (100 mL), and extracted withEtOAc (3×100 mL). The EtOAc solution was washed with brine, dried overanhydrous Na₂SO₄, filtered, concentrated and purified by SiO₂ columnchromatography (6:1 DCM/MeOH) to give compound 678 (98 mg, 53% yield).MS ESI m/z calcd for C₁₀₃H₁₇₀N₁₂O₃₉ [M+H]⁺ 2200.17, found 2200.15.

Example 281. Synthesis of Compound 679

To a solution of compound 678 (98 mg, 0.045 mmol) in dichloromethane (3mL) was added TFA (6 mL). The reaction mixture was stirred at r.t. for 1h, and then concentrated and re-dissolved in DMA (1 mL), compound 41a(31 mg, 0.045 mmol) and DIPEA (12 μL, 0.068 mmol) were added. Thereaction mixture was stirred at r.t. for 90 min, then concentrated andpurified by reverse phase HPLC (C₁₈ column, 10-100% acetonitrile/water)to afford compound 679 (33.6 mg, 30% yield). MS ESI m/z calcd forC₁₁₉H₁₉₄N₁₆O₄₂S [M+H]⁺ 1276.66, found 1276.65.

Example 282. Synthesis of Compound 681

To the solution of compound 301 (1.0 g, 4.3 mmol) and compound 643 (1.6g, 6.4 mmol) in DCM (15 mL) were added HATU (1.83 g, 4.83 mmol) and TEA(0.68 mL, 4.83 mmol) at 0° C. The reaction mixture was allowed to stirat 0° C. for 90 min, then concentrated and purified by columnchromatography (MeOH/DCM) to afford the title compound 681 (2.0 g, >100%yield, containing silica gel). ESI m/z C₂₁H₄₀NO₁₀ [M+H]⁺: 466.26, found466.23.

Example 283. Synthesis of Compound 682

To a solution of compound 681 (2.0 g, 4.3 mmol) in DMF (30 mL) wereadded K₂CO₃ (1.2 g, 8.6 mmol) and BnBr (1.47 g, 8.6 mmol). The mixturewas stirred at r.t. overnight, then poured into a separatory funnelcontaining 100 mL of water and extracted with EtOAc (3×50 mL). Theorganic phase was combined and dried over anhydrous Na₂SO₄, filtered,concentrated and purified by column chromatography (MeOH/DCM) to affordthe title compound 682 (1.0 g, 42% yield). ESI: m/z: calcd forC₂₈H₄₆NO₁₀ [M+H]⁺: 556.30, found 556.30.

Example 284. Synthesis of Compound 683

To a solution of compound 682 (1.0 g, 1.8 mmol) in DCM (1 mL) was addedTFA (3 mL). The reaction was stirred at room temperature for 1 h, thenconcentrated to dryness and co-evaporated twice with DCM, dissolved inDCM (50 mL). Z-Lys-OH (0.5 g, 1.8 mmol), HATU (1.83 g, 4.83 mmol) andTEA (0.68 mL, 4.83 mmol) were added at 0° C. The reaction mixture wasallowed to stir at r.t. for 1 h, then poured into a separatory funnelcontaining 100 mL of water and extracted with 50 mL of DCM three times.The organic phase was combined and washed once with 100 mL of brine,then dried over anhydrous Na₂SO₄, filtered and concentrated. The residuewas purified by column chromatography (MeOH/DCM) to afford the titlecompound 683 (1.0 g, 71% yield). ESI m/z C₃₈H₅₆N₃O₁₃ [M+H]⁺:762.37,found 762.37.

Example 285. Synthesis of Compound 684

To a solution of compound 683 (1.0 g, 1.3 mmol) in DMF (10 mL) was addedHATU (0.60 g, 1.56 mmol), the reaction was stirred at room temperaturefor 30 min. After that, a solution of compound 438 (0.51 g, 1.3 mmol) inDMF (10 mL) and TEA (0.28 mL, 2 mmol) were added at 0° C., and thereaction was stirred at 0° C. for 1 h, then poured into a separatoryfunnel containing 100 mL of water and extracted with EtOAc (50 mL)twice. The organic phases were combined and washed with 100 mL of brine,dried over anhydrous Na₂SO₄, filtered and concentrated. The residue waspurified by column chromatography (MeOH/DCM) to afford the titlecompound 684 (1.20 g, 73% yield). ESI: m/z: calcd for C₆₃H₉₅N₆O₁₈[M+H]⁺: 1223.66, found 1223.66.

Example 286. Synthesis of Compound 685

To a solution of compound 684 (1.20 g, 0.98 mmol) in MeOH (20 mL) wasadded Pd/C (0.1 g, 10 wt %, 50% wet) in a hydrogenation bottle. Themixture was shaken overnight, filtered through Celite (filter aid), andthe filtrate was concentrated and then dissolved in EtOH (50 mL).Compound 125 (0.32 g, 1.16 mmol) and 0.1 M NaH₂PO₄ (10 mL) were addedand the mixture was stirred at r.t. overnight. After concentration, theresidue was purified by column chromatography (MeOH/DCM) to afford thetitle compound 685 (0.57 g, 50% yield). ESI: m/z: caled for C₅₆H₉₀N₇O₁₉[M+H]⁺: 1164.62, found 1164.62.

Example 287. Synthesis of Compound 686

To a solution of compound 685 (0.10 g, 0.086 mmol) in DCM (1 mL) wasadded TFA (3 mL). The reaction was stirred at room temperature for 30min, then concentrated to dryness and co-evaporated twice with DCM, thendissolved in DMA (2 mL) and compound 41a (59 mg, 0.086 mmol) and DIPEA(26 μL, 0.15 mmol) were added. The reaction mixture was stirred at roomtemperature for 1 h and then concentrated, dissolved in 2 mLacetonitrile and purified by reverse phase HPLC with a gradient ofMeCN/H₂O to afforded compound 686 (34 mg, 26% yield). ESI: m/z: calcdfor C₇₂H₁₁₄N₁₁O₂₂S [M+H]⁺: 1516.78, found 1516.78.

Example 288. Synthesis of Compound 688

To a solution of Boc-Glu(OtBu)—OH (0.50 g, 1.65 mmol) in DMF (10 mL)were added HATU (0.69 g, 1.82 mmol) and TEA (0.26 mL, 1.82 mmol). Afterstirring for 30 min, a solution of 11-aminoundecanoic acid (0.33 g, 1.65mmol) in DMF (10 mL) was added and the reaction was stirred at r.t. for1 h, then poured into a separatory funnel containing 200 mL of 1N HCland extracted with DCM (3×50 mL). The organic phase was washed once with100 mL of brine, then dried over anhydrous Na₂SO₄, filtered andconcentrated. The residue was purified by column chromatography(MeOH/DCM) to afford the title compound 688 (1.0 g, >100% yield). ESI:m/z: calcd for C₂₅H₄₇N₂O₇ [M+H]⁺: 487.33, found 487.34.

Example 289. Synthesis of Compound 689

To a solution of compound 688 (1.0 g) in DCM (1 mL) was added TFA (3mL). The reaction was stirred at room temperature for 30 min, thenconcentrated to dryness and dried twice with DCM. Finally, placed on avacuum pump give compound 689 (0.68 g, 2.06 mmol). ESI: m/z: calcd forC₁₆H₃₁N₂O₅ [M+H]⁺: 331.22, found 331.22.

Example 290. Synthesis of Compound 690

To a solution of compound 685 (0.10 g, 0.086 mmol) in EtOAc (6 mL) wasadded pentafluorophenol (18.4 mg, 0.1 mmol) and EDC (19.1 mg, 0.1 mmol).The reaction was stirred at room temperature for 1 h, and then pouredinto a separatory funnel containing 100 mL of water and extracted with50 mL of EtOAc. The organic phase was collected and dried over anhydrousNa₂SO₄, filtered and concentrated. The residue was re-dissolved in 5 mLDMF, and a solution of compound 125 (43 mg, 0.13 mmol) in DMF (5 mL) andDIPEA (35 μL, 0.222 mmol) were added. The reaction was stirred at r.t.for 90 min., then poured into a separatory funnel containing 100 mL of1N HCl and extracted with DCM (2×50 mL). The organic phase was washedonce with 100 mL of brine, then dried over anhydrous Na₂SO₄, filteredand concentrated. The residue was purified by column chromatography(MeOH/DCM) to afford the title compound 690 (0.11 g, 87% yield). ESI:m/z: calcd for C₇₂H₁₁₈N₉O₂₃ [M+H]⁺: 1476.83, found 1476.84.

Example 291. Synthesis of Compound 691

To a solution of compound 691 (0.11 g, 0.074 mmol) in DCM (1 mL) wasadded TFA (3 mL). The reaction was stirred at room temperature for 30min, then concentrated to dryness and co-evaporated twice with DCM, thendissolved in DMA (6 mL). Compound 41a (100 mg, 0.148 mmol) and DIPEA (35μL, 0.222 mmol) were added and the reaction mixture was stirred at roomtemperature for 2 h and concentrated. The residue was dissolved in 2 mLacetonitrile and purified by reverse phase HPLC with a gradient ofMeCN/H₂O to afforded compound 691 (28.6 mg, 21% yield). ESI: m/z: calcdfor C₈₈H₁₄₂N₁₃O₂₆S [M+H]⁺: 1828.98, found 1828.98.

Example 292. General method of Preparation of Conjugate 133, 339, 382,396, 414, 444, 455, 467, 474, 480, 486, 493, 500, 522, 530, 534, 546,550, 556, 560, 564, 574, 584, 593, 601, 613, 619, 626, 637, 641, 650,669, 673, 680, 687, 692, E1, E2, E3, E4, E5, E6 and E7.

To a mixture of 2.0 mL of 10 mg/ml Herceptin in pH 6.0-8.0, were addedof 0.70 ˜ 2.0 mL PBS buffer of 100 mM NaH₂PO₄, pH 6.5-8.5 buffers, TCEP(14-35 μL, 20 mM in water) and the compound 132, mixture of 337 and 338,381, 395, 413, 443, 454, 466, 473, 479, 485, 492, 499, 521, 529, 533,545, 549, 554, 559, 563, 573, 583, 592, 600, 612, 618, 625, 636, 640,649, 668, 672, 679, 686, 691, or maleimide precursor of E1, E2, E3, E4,E5, E6 and E7 (14-28 μL, 20 mM in DMA independently, followed byaddition of 4-(azidomethyl)benzoic acid (14-50 μL, 20 mM in pH 7.5, PBSbuffer). The mixture was incubated at RT for 4-18 h, then DHAA (135 μL,50 mM) was added in. After continuous incubation at RT overnight, themixture was purified on G-25 column eluted with 100 mM NaH₂PO₄, 50 mMNaCl pH 6.0-7.5 buffer to afford 12.2-18.6 mg of the conjugate compound133, 339, 382, 396, 414, 444, 455, 467, 474, 480, 486, 493, 500, 522,530, 534, 546, 550, 556, 560, 564, 574, 584, 593, 601, 613, 619, 626,637, 641, 650, 669, 673, 680, 687, 692, E1, E2, E3, E4, E5, E6 and E7(60%˜93% yield) accordingly in 13.4-15.8 ml of the NaH₂PO₄, buffer. Thedrug/antibody ratio (DAR) was 3.4-4.1 for conjugate, wherein DAR wasdetermined via UPLC-QTOF mass spectrum. It was 94˜99% monomer analyzedby SEC HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID×30 cm, 0.5ml/min, 100 min) and a single band measured by SDS-PAGE gel.

Example 293. In vitro cytotoxicity evaluation of conjugate 133, 339,382, 396, 414, 444, 455, 467, 474, 480, 486, 493, 500, 522, 530, 534,546, 550, 556, 560, 564, 574, 584, 593, 601, 613, 619, 626, 637, 641,650, 669, 673, 680, 687, 692, E1, E2, E3, E4, E5, E6 and E7 incomparison with T-DM1:

The cell line used in the cytotoxicity assays was NCI-N87, a humangastric carcinoma cell line; The cells were grown in RPMI-1640 with 10%FBS. To run the assay, the cells (180 μl, 6000 cells) were added to eachwell in a 96-well plate and incubated for 24 hours at 37° C. with 5%CO₂. Next, the cells were treated with test compounds (20 μl) at variousconcentrations in appropriate cell culture medium (total volume, 0.2mL). The control wells contain cells and the medium but lack the testcompounds. The plates were incubated for 120 hours at 37° C. with 5%CO₂. MTT (5 mg/ml) was then added to the wells (20 μl) and the plateswere incubated for 1.5 hr at 37° C. The medium was carefully removed andDMSO (180 μl) was added afterward. After it was shaken for 15 min, theabsorbance was measured at 490 nm and 570 nm with a reference filter of620 nm. The inhibition % was calculated according to the followingequation: inhibition %=[1-(assay-blank)/(control-blank)]×100. Theresults are listed in Table 1.

TABLE 1 The Structures of the Her2-tubulysin analog conjugates of thepatent application along with their cytotoxicity IC₅₀ results: Con- ju-gate # Structures and its IC50 against NCI-N87 cells 133

339

382

396

414

444

455

467

474

480

486

493

500

522

530

534

546

550

556

560

564

574

584

593

601

613

619

626

637

641

650

669

673

680

687

692

E1

E2

E3

E4

E5

E6

E7

Example 294. Antitumor Activity In vivo (BALB/c Nude Mice BearingNCI-N87 Xenograft Tumor)

The in vivo efficacy of conjugates 474, 486, 493, 601, 626, 637, 641,669, 673, 680, and 692 along with T-DM1 were evaluated in a humangastric carcinoma N-87 cell line tumor xenograft models. Five-week-oldfemale BALB/c Nude mice (78 animals) were inoculated subcutaneously inthe area under the right shoulder with N-87 carcinoma cells (5×10⁶cells/mouse) in 0.1 Ln of serum-free medium. The tumors were grown for 8days to an average size of 140 mm³. The animals were then randomlydivided into 13 groups (6 animals per group). The first group of miceserved as the control group and was treated with the phosphate-bufferedsaline (PBS) vehicle. 12 groups were treated with conjugates 474, 486,493, 601, 626, 637, 641, 669, 673, 680, 692 and T-DM1 respectively atdose of 6 mg/Kg administered intravenously. Three dimensions of thetumor were measured every 3 or 4 days (twice a week) and the tumorvolumes were calculated using the formula tumorvolume=½(length×width×height). The weight of the animals was alsomeasured at the same time. A mouse was sacrificed when any one of thefollowing criteria was met: (1) loss of body weight of more than 20%from pretreatment weight, (2) tumor volume larger than 1500 mm³, (3) toosick to reach food and water, or (4) skin necrosis. A mouse wasconsidered to be tumor-free if no tumor was palpable.

The results were plotted in FIG. 63 . All the 12 conjugates did notcause the animal body weight loss. Here 7 conjugates (673, 637, 650,692, 474, 493 and 486) tested demonstrated better anti-tumor activitythan T-DM1. All 6/6 animals at the groups of compounds 474, 493, and 486had almost no tumor measurable at day 16 till day 28. In contrast T-DM1at dose of 6 mg/Kg was not able to eliminate the tumors and it onlyinhibited the tumor growth for 26 days. In addition, conjugate compounds601, 669, 680, 673, 637, 650, and 692 did not eradicate the tumor atdose of 6 mg/Kg completely. The inhibition of the tumor growth at doseof 6 mg/Kg are:

Conjugate Tumor growth delay T-DM1 26 days 626 <4 days 601 13 days 66918 days 680 25 days 673 >30 days 637 >30 days 650 >30 days 692 >30 days474 >30 days 493 >30 days 486 >30 days

Example 295. Stability study of the conjugates having a sidechain-linkage in comparison with T-DM1 and a regular conjugate (compound133) having a mono-linkage in the mouse serum

Sixty female ICR mice, 6-7 weeks old, were separated into 4 groups. Eachgroup included 15 mice for the PK study of one out of four ADCs. These15 mice were further randomly divided into three groups (n=5). Eachmouse was given conjugates T-DM₁, 133, 680 and 692, respectively at doseof 10 mg/Kg/per rat, i.v. bolus. The blood collection was followed theNCI's Guidelines for Rodent Blood Collection. Basically, mice in eachgroup were taken turn for bleeding in order to avoid more than twicebleedings in a period of 24 hr. Blood was taken from retro-orbital bloodsinus with a 70 uL capillary at time 0 (pre-dosing), 0.083, 0.25, 0.5,1, 4, 8, 24, 48, 96, 168, 312 and 504 hrs post dosing. Plasma sampleswere analyzed for total antibodies and drug-conjugated antibodies byspecific ELISA techniques. In brief, the conjugated antibody or thetotal antibody concentration in the mouse serum was measured as follows:96-well ELISA plates were respectively coated overnight at 4° C. withanti-DM1 antibody, anti-tubulysin antibody or anti-Her-2's Fab antibody(1 ug/mL in 10 mM PBS, pH7.2). The plates were then washed three timeswith a washing buffer PBS-T (PBS/0.02% Tween20), and then blocked with adilution buffer 1% (w/v) BSA/PBS-T for 1 hour at 37° C. After theblocking buffer was removed, the standards or mouse serum samples eachwith triple replicates were diluted in 1% BSA/PBS-T buffer, incubated at37° C. for 1 hour, then the AP-conjugated donkey anti-human antibody wasadded for 30 minutes at 37° C. after the plates were washed. Plates werewashed again, followed by the addition of pNPP substrate for the colordevelopment and then read on a microplate reader at 405 nm wavelengthonce the color development reaction was quenched with the 1 mol/L sodiumhydroxide. The concentration of the conjugated antibody or the totalantibody was obtained from a four-parameter curve fitting of thestandard curve.

The result as shown in FIG. 64 , the PK behaviors of total antibodiesand drug-conjugated antibodies after dosing four ADCs presented astypical two-phase clearance curves. Equivalences between plasma andperipheral tissues were reached 8 hrs post-dosing. Elimination phaseemerged 24 hr post-dosing and continued until the last sampling timepoint. In summary, the values of conjugate exposures (Auciast) for thesethree ADCs are 14981, 14857, 17212 and 17638 hr ug/kg for T-DM1, 133,680 and 692 respectively. Distribution volumes for all these threeconjugates are double of total blood volumes. The clearances (CL) of theconjugates are 0.59, 0.59, 0.48, and 0.45 mL/hr/kg, which are almosthalves of those for total antibodies. The clearance of 692 and 680 boththeir conjugates and total antibodies, are smaller than T-DM1 and theregular conjugate 133, which indicates that the conjugates having thebranched-linkage are more stable than the regular mono-linked conjugatesin the mouse serum.

Example 296. Liver toxicity Study of the conjugate having abranched-linkage in comparison with regular conjugates (compound 133 andT-DM1) having a regular linkage. Eight-four female ICR mice, 6-7 weeksold, were separated into 14 groups. Each group included 6 mice for theliver toxicity study. The first group of mice served as the controlgroup and was treated with the phosphate-buffered saline (PBS) vehicle.13 groups were treated with conjugates 133, 474, 486, 493, 601, 626,637, 641, 669, 673, 680, 692 and T-DM1 respectively at dose of 200 mg/Kgadministered intravenously. The blood collection was followed the NCI'sGuidelines for Rodent Blood Collection. Basically, Blood samples werecollected through retro-orbital sinuses of the mice, and centrifuged toobtain the sera on Day 5 and 12 after administration. The levels ofaspartate aminotransferase (AST), alanine aminotransferase (ALT) andalkaline phosphatase (ALP) were analyzed using PUS-2018 semi-automaticbiochemistry analyzer with a commercial kid (using aspartate and alanineas substrates, respectively). Reference values were established byfollowing reactive dynamics, according to manufacturer'srecommendations. The results on average are show in Table 2 below

AST (IU/mL) ALT(IU/mL) ALP(IU/mL) Compound Day 5 Day 12 Day 5 Day 12 Day5 Day 12 PBS  91  95  46  36 186 179 T-DM1 3276 1509 412 453 495 502 1333683 1762 461 523 498 382 474 1283  276 184 125 288 228 486 1873 1539201 263 381 301 493 1521  602 197 165 323 287 601  111  105  86  46 206189 626  151  108  96  49 197 193 637  851  178 186  79 267 187 641  918 183 186  87 287 172 650  832  173 186  78 279 167 673  653  158 136  59207 181 680  193  98  76  39 256 189 692  238  91 106  37 273 189

The liver toxicity results indicate that at the much higher dose of 150mg/Kg the conjugates (474, 486, 493, 601, 626, 637, 641, 669, 673, 680,and 692) with the side chain linker are much less toxic than both T-DM1and the regular mono-linked conjugate 133. Since conjugates 474, 486,493, 637, 641, 669, 673, 680, and 692 have better in vivo activity thanT-DM1, therefore the overall therapeutical windows for conjugates 474,486, 493, 637, 641, 669, 673, 680, and 692 would be much wider thanT-DM1.

1. A side chain-linkage compound of Formula (IV), which can readilyreact to a cell-binding molecule T to form a conjugate:

wherein “

” represents a single bond; L₁ and L₂ are, the same or different,independently selected from O, NH, N, S, P, NNH, NHNH, N(R₃),N(R₃)N(R₃′), CH, CO, C(O)NH, C(O)O, NHC(O)NH, NHC(O)O, polyethyleneoxyunit of formula (OCH₂CH₂)_(p)OR₃, or (OCH₂CH—(CH₃))_(p)OR₃, orNH(CH₂CH₂O)_(p)R₃, or NH(CH₂CH(CH₃)O)_(p)R₃, or N[(CH₂CH₂O)_(p)R₃],[(CH₂CH₂O)_(p)R₃′], or (OCH₂CH₂)_(p)COOR₃, or CH₂CH₂(OCH₂CH₂)_(p)COOR₃,wherein p and p′ are independently an integer selected from 0 to about1000, or a combination of two or more thereof; C₁-C₈ alkyl; C₂-C₈heteroalkyl, alkylcycloalkyl, or heterocycloalkyl; C₃-C₈ aryl, Ar-alkyl,heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl,alkylcarbonyl, or heteroaryl; or (Aa)_(r), r=1-12 (1 to 12 amino acidunits), which is composed from natural or unnatural amino acids, or thesame or different sequences of dipeptide, tripeptide, tetrapeptide,pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide,decapeptide, undecapeptide or dodecapeptide unit; W is a stretcher unithaving C₁-C₁₈, normally a self-immolative spacer, a peptidic unit, ahydrazone, a disulfide, a thioether, an ester, or an amide bond; w is 1or 2 or 3; V₁ and V₂ are independently a spacer unit and selected fromO, NH, S, C₁-C₈ alkyl, C₂-C₈ heteroalkyl, alkenyl, or alkynyl, C₃-C₈aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, oralkylcarbonyl, or (Aa)_(r), r=1-12 (1 to 12 amino acid units), which iscomposed from a natural or unnatural amino acid, or the same ordifferent sequences of dipeptide, tripeptide, tetrapeptide,pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide,decapeptide, undecapeptide or dodecapeptide unit; or (CH₂CH₂O)_(p), p is0-1000; and v₁ and v₂ are independently 0, 1 or 2, but v₁ and v₂ are not0 at the same time; when v₁ or v₂ is 0, one of the side chain Q₁ or Q₂fragment is absent; Q₁ and Q₂ are independently represented by Formula(I-q1):

wherein

is a site linked to L₁ or L₂; G₁ and G₂ are independently OC(O), NHC(O),C(O), CH₂, NH, OC(O)NH, NHC(O)NH, O, S, B, P(O)(OH), NHP(O)(OH),NHP(O)(OH)NH, CH₂P(O)(OH)NH, OP(O)(OH)O, CH₂P(O)(OH)O, NHS(O)₂,NHS(O)₂NH, CH₂S(O)₂NH, OS(O)₂O, CH₂S(O)₂O, Ar, ArCH₂, ArO, ArNH, ArS,ArNR₁, or (Aa)_(q1); G₃ is OH, SH, OR₁, SR₁, OC(O)R₁, NHC(O)R₁, C(O)R₁,CH₃, NH₂, NR₁, +NH(R₁), +N(R₁)(R₂), C(O)OH, C(O)NH₂, NHC(O)NH₂, BH₂,BR₁R₂, P(O)(OH)₂, NHP(O)(OH)₂, NHP(O)(NH₂)₂, S(O)₂(OH),(CH₂)_(q1)C(O)OH, (CH₂)_(q1)P(O)(OH)₂, C(O)(CH₂)_(q1)C(O)OH,OC(O)(CH₂)_(q1)C(O)OH, NHC(O)(CH₂)_(q1)C(O)OH, CO(CH₂)_(q1)P(O)(OH)₂,NHC(O)O(CH₂)_(q1)C(O)OH, OC(O)NH(CH₂)_(q1)C(O)OH,NHCO(CH₂)_(q1)P(O)(OH)₂, NHC(O)(NH)(CH₂)_(q1)C(O)OH,CONH(CH₂)_(q1)P(O)(OH)₂, NHS(O)₂(CH₂)_(q1)C(O)OH, CO(CH₂)_(q1)S(O)₂(OH),NHS(O)₂NH(CH₂)_(q1)C(O)OH, OS(O)₂NH(CH₂)_(q1)C(O)OH,NHCO(CH₂)_(q1)S(O)₂(OH), NHP(O)(OH)(NH)(CH₂)_(q1)C(O)OH,CONH(CH₂)_(q1)S(O)(OH), OP(O)(OH)₂, (CH₂)_(q1)P(O)(NH)₂, NHS(O)₂(OH),NHS(O)₂NH₂, CH₂S(O)₂NH₂, OS(O)₂OH, OS(O)₂OR₁, CH₂S(O)₂OR₁, Ar, ArR₁,ArOH, ArNH₂, ArSH, ArNHR₁, or (Aa)_(q1); (Aa)_(q1) is a peptidecontaining the same or different sequence of natural or unnatural aminoacids; X₁ and X₂ are independently O, CH₂, S, S(O), NHNH, NH, N(R₁),+NH(R₁), +N(R₁)(R₂), C(O), OC(O), OC(O)O, OC(O)NH, or NHC(O)NH; Y₂ is O,NH, NR₁, CH₂, S, NHNH, or Ar; p₁, p₂ and p₃ are independently 0-100 butare not 0 at the same time; q₁ and q₂ are independently 0-24; R₁, R₂, R₃and R₃′ are independently H, C₁-C₈ alkyl; C₂-C₈ heteroalkyl, orheterocyclic; C₃-C₈ aryl, Ar-alkyl, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroalkylcycloalkyl, carbocyclic, or alkylcarbonyl;alternatively, any one or more of W, Q₁, Q₂, L₁, L₂, V₁, or V₂ can beindependently absent but Q₁ and Q₂ are not absent at the same time; D istubulysin analog having following formula (II):

or a pharmaceutically acceptable salt, hydrate, or hydrated salt; or apolymorphic crystalline structure; or an optical isomer, racemate,diastereomer or enantiomer thereof, wherein ----- is a linkage site thatlinks to W independently; wherein R¹, R₂, R³, and R⁴ are independentlyH, C₁-C₈ alkyl; C₂-C₈ heteroalkyl, or heterocyclic; C₃-C₈ aryl,Ar-alkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl,heteroalkylcycloalkyl, carbocyclic, or alkylcarbonyl; or R¹ and R₂, R¹and R³, R² and R³, R³ and R⁴, R⁵ and R⁶, R¹¹ and R¹², or R¹³ and R¹⁴form a 3-7 membered carbocyclic, cycloalkyl, heterocyclic,heterocycloalkyl, aromatic or heteroaromatic ring system; R¹ and R² canbe independently absent when they link to W independently orsimultaneously, Y¹ is N or CH; wherein R⁵, R⁶, R⁸, R¹⁰ and R¹¹ areindependently H, or C₁-C₄ alkyl or heteroalkyl; wherein R⁷ isindependently H, R¹⁴, —R¹⁴C(═O)X¹R¹⁵; or —R¹⁴X¹R¹⁵; X¹ is O, S, S—S, NH,CH₂ or NR¹⁴; wherein R⁹ is selected from H, OH, —O—, ═O, —OR¹⁴,—OC(═O)R¹⁴, —OC(═O)NHR¹⁴—, —OC(═O)R¹⁴SSR¹⁵—, OP(═O)(OR¹⁴)—,—OC(═O)NR¹⁴R¹⁵, OP(═O)(OR¹⁴), or OR¹⁴0P(═O)(OR¹⁵); wherein R¹¹ isindependently H, R¹⁴, —R¹⁴C(═O)R¹⁶, —R¹⁴X²R¹⁶, —R¹⁴C(═O)X², wherein X²is —O—, —S—, —NH—, —N(R¹⁴)—, —O—R¹⁴—, —S—R¹⁴—, —S(═O)—R¹⁴—, or —NHR¹⁴;wherein R¹² is R¹⁵, —OH, —SH, —NH₂, NH, NHNH₂, —NH(R¹⁵), —OR¹⁵,—R¹⁵COR¹⁶, R¹⁵COOR¹⁶, —R¹⁵C(O)NH₂, —R¹⁵C(O)NHR¹⁷, —SR¹⁶, R¹⁵S(═O)R¹⁶,—R¹⁵P(═O)(OR¹⁷)₂, —R¹⁵OP(═O)(OR¹⁷)₂, —CH₂OP(═O)(OR¹⁷)₂, —R¹⁵SO₂R¹⁷,—R¹⁵X²R¹⁶, —R¹⁵C(═O)X², where X² is —O—OH, SH, —S—, NH₂, —NH—, —N(R¹⁵)—,—O—R¹⁵—, —S—R¹⁵—, —S(═O)—R¹⁵—, CH₂ or —NHR⁵—; R¹³ and R¹⁴ areindependently H, O, S, NH, N(R¹⁵), NHNH, —OH, —SH, —NH₂, NH, NHNH₂,—NH(R¹⁵), —OR¹⁵, CO, —COX², —COX²R¹⁶, R¹⁷, F, Cl, Br, I, SR¹⁶, NR¹⁶R¹⁷,N═NR¹⁶, N═R¹⁶, NO₂, SOR¹⁶R¹⁷, SO₂R¹⁶, SO₃R¹⁶, PR¹⁶, PR¹⁶R¹⁷, POR¹⁶R¹⁷,PO₂R¹⁶R¹⁷, OP(O)(OR¹⁷)₂, OCH₂OP(O)(OR¹⁷)₂, OC(O)R¹⁷, OC(O)OP(O)(OR¹⁷)₂,PO(OR¹⁶)(OR¹⁷), OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂, OC(O)NHR¹⁷, —O—(C₄-C₁₂glycoside), —N—(C₄-C₁₂ glycoside); C₁-C₈ alkyl or heteroalkyl; C₂-C₈alkenyl, alkynyl, heteroalkyl, or heterocycloalkyl; C₃-C₈ aryl,Ar-alkyl, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl,or heteroaryl, or C₂-C₈ ester, ether, or amide; or peptide containing1-8 amino acids (NH(Aa)₁₋₈ or CO(Aa)i-s (N-terminal or C-terminal 1-8same or different amino acids), or polyethyleneoxy unit of formula(OCH₂CH₂)_(p) or (OCH₂CH(CH₃))_(p), wherein p is an integer from 0 toabout 1000, or a combination of two or more thereof; X² is O, S, S—S,NH, CH₂, OH, SH, NH₂, CHR¹⁴ or NR¹⁴; R¹⁵, R¹⁶ and R¹⁷ are independentlyH, C₁-C₈ alkyl or heteroalkyl; C₂-C₈ alkenyl, alkynyl, heteroalkyl, orheterocycloalkyl; C₃-C₈ aryl, Ar-alkyl, carbocyclic, cycloalkyl,heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, or alkylcarbonyl, orNa⁺, K⁺, Cs⁺, Li⁺, Ca²⁺, Mg⁺, Zn²⁺, N⁺(R¹)(R²)(R³)(R⁴), or HN⁺(C₂H₅OH)₃salt; Y¹ and Y² are independently N or CH; q is 0 or 1; when q=0, Y³does not exist, Y⁴, Y⁵, Y⁶ and Y⁷ are independently CH, N, NH, O, S, orN(R′), thus Y², Y⁴, YS, Y⁶ and Y⁷form a heteroaromatic ring of furan,pyrrole thiophene, thiazole, oxazole, imidazole, pyrazole, triazole,tetrazole, or thiadiazole; when q=1, Y³, Y⁴, Y⁵, Y⁶ and Y⁷ areindependently CH or N, thus Y², Y³, Y⁴, Y⁵, Y⁶ and Y⁷ form an aromaticring of benzene, pyridine, pyridazine, pyrimidine, pyrazine, triazine,tetrazine, or pentazine; L_(V1) is a reacting group that can be reactedwith a thiol, amine, carboxylic acid, selenol, phenol or hydroxyl groupon a cell-binding molecule; L_(V1) is selected from OH; F; Cl; Br; I;nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol;pentafluorophenol; tetrafluorophenol; difluorophenol; mono-fluorophenol;pentachlorophenol; triflate; imidazole; dichlorophenol;tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate;2-ethyl-5-phenylisoxazolium-3′-sulfonate, anhydride formed by an aciditself, or formed with another anhydride; or an intermediate moleculegenerated with a condensation reagent for peptide coupling reactions, orfor Mitsunobu reactions; the condensation reagent is selected from: EDC(N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide), DCC(dicyclohexyl-carbodiimide), N,N′-diisopropylcarbodiimide (DIC),N-cyclohexyl-N′-(2-morpholino-ethyl)carbodiimidemetho-p-toluenesulfonate (CMC, or CME-CDI), 1,1′-carbonyldiimi-dazole(CDI), TBTU (0-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate),N,N,N′,N′-tetramethyl-O-(1H-benzo-triazol-1-yl)-uroniumhexafluoro-phosphate (HBTU),(benzotriazol-1-yloxy)tris(dimethylamino)-phosphoniumhexafluorophosphate (BOP),(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate(PyBOP), diethyl cyanophosphonate (DEPC),chloro-N,N,N′,N′-tetra-methylformamidiniumhexafluorophosphate,1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU),1-[(dimethylamino)-(morpholino)methylene]-1H-[1,2,3]triazolo[4,5-b]pyridine-1-ium3-oxide hexafluoro-phosphate (HDMA),2-chloro-1,3-dimethyl-imidazolidinium hexafluorophosphate (CIP),chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP),fluoro-N,N,N′,N′-bis(tetramethylene)-formamidinium hexafluorophosphate(BTFFH), N,N,N′,N′-tetramethyl-S-(1-oxido-2-pyridyl)thiuroniumhexafluorophosphate, O-(2-oxo-1(2H)pyridyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TPTU),S-(1-oxido-2-pyridyl)-N,N,N′,N′-tetramethylthiuronium tetrafluoroborate,O-[(ethoxycarbonyl)-cyanomethylenamino]-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU),O-(benzotriazol-1-yl)-N,N,N′,N′-bis(tetramethylene)-uroniumhexafluorophosphate (HBPyU), N-benzyl-N′-cyclohexyl-carbodiimide (with,or without polymer-bound), dipyrrolidino(N-succinimidyl-oxy)carbeniumhexafluoro-phosphate (HSPyU), chlorodipyrrolidinocarbeniumhexafluorophosphate (PyClU), 2-chloro-1,3-dimethylimidazoli-diniumtetrafluoroborate(CIB), (benzotriazol-1-yloxy)dipiperidino-carbeniumhexafluorophosphate (HBPipU),0-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TCTU), bromotris(dimethylamino)-phosphoniumhexafluorophosphate (BroP), propylphosphonic anhydride (PPACA, T3P©),2-morpholinoethyl isocyanide (MEI),N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium hexafluorophosphate(HSTU), 2-bromo-1-ethyl-pyridinium tetrafluoroborate (BEP),O-[(ethoxycarbonyl)cyano-methylenamino]-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TOTU),4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholiniumchloride (MMTM,DMTMM), N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uroniumtetrafluoroborate (TSTU),O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoro-borate (TDBTU),1,1′-(azodicarbonyl)-dipiperidine (ADD),di-(4-chlorobenzyl)azodicarboxylate (DCAD), di-tert-butylazodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD), diethylazodicarboxylate (DEAD); L_(V1) is also an anhydride, formed by an aciditself or formed with another C₁-C₈ acid anhydride.
 2. The sidechain-linkage compound according to claim 1, wherein L_(V1) is selectedfrom:

wherein X₁′ is F, Cl, Br, I or L_(v3); X₂′ is O, NH, N(R₁), or CH₂; R₃is independently H, aromatic, heteroaromatic, or aromatic group whereinone or several H atoms are replaced independently by —R₁,-halogen, —OR₁,—SR₁, —NR₁R₂, — NO₂, —S(O)R₁, —S(O)₂R₁, or —COOR₁; L_(v3) is a leavinggroup selected from F, Cl, Br, I, nitrophenol; N-hydroxysuccinimide(NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol;difluorophenol; monofluorophenol; pentachlorophenol; triflate;imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole;tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3′-sulfonate, anhydrideformed by an acid itself, or formed with another anhydride: acetylanhydride, formyl anhydride; or an intermediate molecule generated witha condensation reagent for peptide coupling reactions or for Mitsunobureactions.
 3. The side chain-linkage compound according to claim 1,wherein Q₁ and Q₂ are independently a C₂-C₁₀₀ polycarboxylacid; aC₂-C₁₀₀ polyalkylamine; a C₆-C₁₀₀ oligosaccharide or polysaccharide; aC₆-C₁₀₀ zwitterionic betaine or zwitterionic poly(sulfobetaine)) (PSB)that consists of a quaternary ammonium cation and a sulfonate anion; aC₆-C₁₀₀ biodegradable polymer composed of poly(lactic/glycolic acid)(PLGA), poly(acrylates), chitosan, copolymer ofN-(2-hydroxypropyl)methacrylamide, poly[2-(methacryloyloxy)ethylphosphorylcholine](PMPC), poly-L-glutamic acid,poly(lactide-co-glycolide) (PLG), poly(lactide-co-glycolide),poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG),poly(lactide-co-glycolide), poly(ethylene glycol)-modified peptide,poly(ethylene glycol)-containing an amino acid or peptide, poly(ethyleneglycol)-modified lipid, poly(ethylene glycol)-modified alkylcarboxicacid, poly(ethylene glycol)-modified alkylamine,poly(lactide-co-glycolide, hyaluronic acid (HA) (glycosaminoglycan),heparin/heparan sulfate (HSGAGs), chondroitin sulfate/dermatan sulfate(CSGAGs), poly(ethylene glycol)-modified alkylsulfate, poly(ethyleneglycol)-modified alkylphosphate, or poly(ethylene glycol)-modified alkylquaternary ammonium.
 4. The side chain-linkaged compound according toclaim 1, wherein Q₁ and Q₂ are independently selected from Iq-01 toIq-35:

wherein R₂₅ and R₂₅′ are independently selected from H; HC(O), CH₃C(O),CH₃C(NH), C₁-C₁₈ alkyl, C₁-C₁₈ alkyl, alkyl-Y₁—SO₃H, C₁-C₁₈alkyl-Y₁—PO₃H₂, C₁-C₁₈ alkyl-Y₁—CO₂H, C₁-C₁₈ alkyl-Y₁—N⁺R₁′R₂′R₃′R⁴′,C₁-C₁₈ alkyl-Y₁—CONH₂, C₂-C₁₈ alkylene, C₂-C₁₈ ester, C₂-C₁₈ ether,C₂-C₁₈ amine, C₂-C₁₈ alkyl carboxylamide, C₃-C₁₈ Aryl, C₃-C₁₈ cyclicalkyl, C₃-C₁₈ heterocyclic, 1-24 amino acids; C₂-C₁₈ lipid, a C₂-C₁₈fatty acid or a C₂-C₁₈ fatty ammonium lipid; X₁ and X₂ are independentlyselected from NH, N(R₁′), O, CH₂, S, C(O), S(O), S(O₂), P(O)(OH), NHNH,CH═CH, Ar or (Aa)_(q1), q₁=0-24 (0-24 amino acids, q₁=0 means absent);X₁, X₂, X₃, X₄, Y₁, Y₂ and Y₃ are independently selected from NH,N(R₁′), O, C(O), CH₂, S, S(O), NHNH, C(O), OC(O), OC(O)O, OC(O)NH,NHC(O)NH, Ar or (Aa)_(q1), X₁, X₂, X₃, X₄, Y₁, Y₂ and Y₃ can beindependently absent; p₁, p₂ and p₃ are independently 0-100 but are not0 at the same time; q₁, q₂ and q₃ are independently 0-24; R₁′, R₂′, R₃′and R⁴′ are independently selected from H and C₁-C₆ alkyl; Aa is naturalor unnatural amino acid; Ar or (Aa)_(q1), is the same or differentsequence of peptides; q₁=0 means (Aa)_(q1) absent.
 5. The sidechain-linkaged compound according to claim 1, wherein D (tubulysinstructure) is selected from 1-01 to 1-75:

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers; wherein R²⁰ isH; C₁-C₈ linear or branched alkyl or heteroalkyl; C₂-C₈ linear orbranched alkenyl, alkynyl, alkylcycloalkyl, or heterocycloalkyl; C₃-C₈linear or branched aryl, Ar-alkyl, heterocyclic, carbocyclic,cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl;carbonate (—C(O)OR¹⁷), carbamate (—C(O)NR¹⁷R¹⁸); or C₁-C₈ carboxylate,ester, ether, or amide; or 1-8 amino acids; or polyethyleneoxy unit offormula (OCH₂CH₂)_(p) or (OCH₂CH(CH₃))_(p), wherein p is an integer from0 to about 1000; or R²⁰ is absent and the oxygen atom forms a ketone, ora combination of two of more thereof, Z² and Z³ are independently H, OH,NH₂, O, NH, COOH, COO, C(O), C(O), C(O)NH, C(O)NH₂, R¹⁸,OCH₂OP(O)(OR¹8)₂, OC(O)OP(O)(OR¹8)₂, OPO(OR¹8)₂, NHPO(OR¹8)₂,OP(O)(OR¹8)OP(O)(OR¹8)₂, OC(O)R¹8, OC(O)NHR¹8, OSO₂(OR¹8),O—(C₄-C₁₂-glycoside), C₁-C₈ linear or branched alkyl or heteroalkyl;C₂-C₈ linear or branched alkenyl, alkynyl, alkylcycloalkyl, orheterocycloalkyl; C₃-C₈ linear or branched aryl, Ar-alkyl, heterocyclic,carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, orheteroaryl; carbonate (—C(O)OR¹⁷), carbamate (—C(O)NR¹⁷R¹⁸); R¹⁷ and R¹⁸are independently H, C₁-C₈ linear or branched alkyl or heteroalkyl;C₂-C₈ linear or branched alkenyl, alkynyl, alkylcycloalkyl, orheterocycloalkyl; C₃-C₈ linear or branched aryl, Ar-alkyl, heterocyclic,carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, orheteroaryl; carbonate (—C(O)OR¹⁷), carbamate (—C(O)NR¹⁷R¹⁸); R¹⁹ is H,OH, NH₂, OSO₂(OR¹⁸), XCH₂OP(O)(OR¹⁸)₂, XPO(OR¹⁸)₂, XC(O)OP(O)(OR¹⁸)₂,XC(O)R¹⁸, XC(O)NHR¹⁸, C₁-C₈ alkyl or carboxylate; C₂-C₈ alkenyl,alkynyl, alkylcycloalkyl, or heterocycloalkyl; C₃-C₅ aryl oralkylcarbonyl; or pharmaceutical salts; X is O, S, NH, NHNH, or CH₂; R⁷is defined the same as in claim
 1. 6. The side chain-linkaged compoundaccording to claim 1, wherein W, L₁, L₂, V₁, and V₂ independently iscomposed of one or more linker components of the following structures:

or L- or D-, natural or unnatural peptides containing 1-20 the same ordifferent amino acids;

wherein

is a site of linkage; X₂, X₃, X₄, X₅, and X₆ are independently selectedfrom NH; NHNH; N(R₃); N(R₃)N(R₃′); 0; S; C₁-C₆ alkyl; C₂-C₆ heteroalkyl,alkylcycloalkyl, or heterocycloalkyl; C₃-C₈ aryl, Ar-alkyl,heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl,alkylcarbonyl, or heteroaryl; or 1-8 amino acids; wherein R₃ and R₃′ areindependently H; C₁-C₈ alkyl; C₂-C₈ hetero-alkyl, alkylcycloalkyl, orheterocycloalkyl; C₃-C₈ aryl, Ar-alkyl, heterocyclic, carbocyclic,cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; orC₁-C₈ ester, ether, or amide; or polyethyleneoxy unit of formula(OCH₂CH₂)_(p) or (OCH₂CH(CH₃))_(p), wherein p is an integer from 0 toabout 1000, or a combination of two or more thereof.
 7. The sidechain-linkaged conjugate compound according to claim 1, wherein W, L₁,L₂, V₁, and V₂ independently is composed of: (A): a self-immolativecomponent, peptidic units, a hydrazone bond, a disulfide, an ester, anoxime, an amide, or a thioether bond; the self-immolative unit includesaromatic compounds that are electronically similar topara-aminobenzyl-carbamoyl (PAB) groups, 2-aminoimidazol-5-methanolderivatives, heterocyclic PAB analogs, beta-glucuronide, and ortho orpara-aminobenzylacetals; or one of the following structures:

wherein the (*) atom is a point of attachment of another component; X₁,Y₁, Z² and Z³ are independently NH, O, or S; Z¹ is independently H,NHR₁, OR₁, SR₁, or COX₁R₁, wherein X₁ and Ri are defined the same as inclaim 1; v is 0 or 1; U¹ is independently H, OH, C₁-C₆ alkyl,(OCH₂CH₂)_(n), F, Cl, Br, I, OR₅, SR₅, NR₅R₅′, N═NRs, N═R₅, NR₅R₅′, NO₂,SOR₅R₅′, SO₂R₅, SO₃R₅, OSO₃R₅, PR₅R₅′, POR₅R₅′, PO₂R₅R₅′,OPO(OR₅)(OR₅′), or OCH₂PO(OR₅(OR₅′), wherein R₅ and R₅′ areindependently selected from H, C₁-C₈ alkyl; C₂-C₈ alkenyl, alkynyl,heteroalkyl, or amino acid; C₃-C₈ aryl, heterocyclic, carbocyclic,cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl, orglycoside; or pharmaceutical cation salts thereof; (B): anon-self-immolative linker component containing one of the followingstructures:

wherein the (*) atom is a site of attachment; X¹, Y¹, U¹, R₅, R₅′ aredefined as above; r is 0-100; m and n are 0-20 independently; (C): areleasable component that at least one bond that can be broken underphysiological conditions: a pH-labile, acid-labile, base-labile,oxidatively labile, metabolically labile, biochemically labile orenzyme-labile bond, which having one of the following structures:—(CR₅R₆)_(m)(Aa)_(r)(CR₇R₈)_(n)(OCH₂CH₂)_(t)—,—(CR₅R₆)_(m)(CR₇R₈)_(n)(Aa)_(r)(OCH₂CH₂)_(t)—,—(Aa)_(r)—(CR₅R₆)_(m)(CR₇R₈)_(n)(OCH₂CH₂)_(t)—,—(CR₅R₆)_(m)(CR₇R₈)_(n)(OCH₂CH₂)_(r)(Aa)_(t)—,—(CR₅R₆)_(m)—(CR₇═CR₅)(CR₉R₁₀)_(n)(Aa)_(t)(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)(NR₁₁CO)(Aa)_(t)(CR₉R₁₀)_(n)—(OCH₂CH₂)_(r)—(CR₅R₆)_(m)(Aa)_(t)(NR₁₁CO)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)(OCO)(Aa)_(t)(CR₉R₁₀)_(n)—(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)(OCNR₇)(Aa)_(t)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)(CO)(Aa)_(t)—(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—(CR₅R₆)_(m)(NR₁₁CO)(Aa)_(t)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)—(OCO)(Aa)_(t)(CR₉R₁₀)_(n)—(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)(OCNR₇)(Aa)_(t)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)(CO)(Aa)_(t)(CR₉R₁₀)_(n)—(OCH₂CH₂)_(r)—,—(CR₅R₆)_(m)-phenyl-CO(Aa)_(t)(CR₇R₈)_(n)—,—(CR₅R₆)_(m)-furyl-CO(Aa)_(t)(CR₇R₈)_(n)—,—(CR₅R₆)_(m)—oxazolyl-CO(Aa)_(t)(CR₇R₈)_(n)—,—(CR₅R₆)_(m)-thiazolyl-CO(Aa)_(t)(CCR₇R₈)_(n)—,—(CR₅R₆)_(t)-thienyl-CO(CR₇R₈)_(n)—,—(CR₅R₆)_(t)-inidazolyl-CO—(CR₇R₈)_(n)—,—(CR₅R₆)_(t)-morpholino-CO(Aa)_(t)—(CR₇R₈)_(n)—,—(CR₅R₆)_(t)piperazino-CO(Aa)_(t)—(CR₇R₈)_(n)—,—(CR₅R₆)_(t)—N-methylpiperazin-CO(Aa)_(t)—(CR₇R₈)_(n)—,—(CR₅R)_(m)—(Aa)_(t)phenyl-, —(CR₅R₆)_(m)—(Aa)_(t)furyl-,—(CR₅R₆)_(m)-oxazolyl(Aa)_(r)—, —(CR₅R₆)_(m)-thiazolyl(Aa)_(t)—,—(CR₅R₆)_(m)-thienyl-(Aa)_(t)—, —(CR₅R( )_(m)-imidazolyl(Aa)_(t)—,—(CR₅R₆)_(m)-morpholino-(Aa)_(t)—, —(CR₅R₆)_(m)-piperazino-(Aa)_(t)—,—(CR₅R₆)_(m)—N-methylpiperazino-(Aa)_(t)—,—K(CR₅R₆)_(m)(Aa)_(r)(CR₇R₈)_(n)(OCH₂CH₂)_(t)—,—K(CR₅R₆)_(m)(CR₇R₈)_(n)—(Aa)_(r)(OCH₂CH₂)_(t)—,—K(Aa)_(r)—(CR₅R₆)_(m)(CR₇R₈)_(n)(OCH₂CH₂)_(t)—,—K(CR₅R₆)_(m)—(CR₇R₈)_(n)(OCH₂—CH₂)_(r)(Aa)_(t)—,—K(CR₅R₆)_(m)—(CR₇═CR₈)(CR₉R₁₀)_(n)(Aa)_(t)(OCH₂CH₂)_(r)—,—K(CR₅R₆)_(m)—(NR₁₁CO—)(Aa)_(t)(CR₉R₁₀)_(n)(OCH₂C₁₂)_(r)—,—K(CR₅R₆)_(m)(Aa)_(t)(NR₁₁CO)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₅R₆)_(m)(OCO)(Aa)_(t)(CR₉R₁₀)_(n)—(OCH₂CH₂)_(r)—,—K(CR₅R₆)_(m)(OCNR₇)(Aa)_(t)(CR₉R₁₀)_(n)—(OCH₂CH₂)_(r)—,—K(CR₅R₆)_(m)(CO)(Aa)_(t)—(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₅R₆)_(m)(NR₁₁CO)(Aa)_(t)—(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₅R₆)_(m)—(OCO)(Aa)_(t)(CR₅R₁₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₅R₆)_(m)—(OCNR₇)(Aa)_(t)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—K—(CR₅R₆)_(m)(CO)(Aa)_(t)(CR₉R₁₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₅R₆)_(m)-phenyl—CO(Aa)_(t)(CR₇R₈)_(n)—,—K—(CR₅R₆)_(m)-furyl-CO(Aa)_(t)—(CR₇R₈)_(n)—,—K(CR₅R₆)_(m)-oxazolyl-CO(Aa)_(t)—(CR₇R₈)_(n)—, —K(CR₅R₆)_(m)-thiazolyl-CO(Aa)_(t)—(CR₇R₈)_(n)—, —K(CR₅R₆)_(t)-thienyl-CO(CR₅R₈)_(n)—,—K(CR₅R₆)_(t)imidazolyl-CO—(CR₇R₈)_(n)—,—K(CR₅R₆)_(t)-morpholino-CO—(Aa)_(t)(CR₇R₈)_(n)—,—K(CR₅R₆)_(t)-piperazino-CO(Aa)_(t)—(CR₇R₈)_(n)—,—K(CR₅R₆)_(t)—N-methylpiperazin-CO(Aa)_(t)(CR₇R₈)_(n)—,—K(CR₅R)_(m)—(Aa)_(t)phenyl, —K—(CR₅R₆)_(m)—(Aa)_(t)furyl-,—K(CR₅R₆)_(m)-oxazolyl(Aa)_(t)—, —K(CR₅R₆)_(m)-thiazolyl(Aa)_(t)—,—K(CR₅R₆)_(m)-thienyl-(Aa)_(t)—, —K(CR₅R₆)_(m)-imidazolyl(Aa)_(t)—,—K(CR₅R₆)_(m)-morpholino(Aa)_(t)—, —K(CR₅R₆)_(m)-piperazino-(Aa)_(t)—,—K(CR₅R₆)_(m)—N-methylpiperazino(Aa)_(t)—; wherein Aa, m, and n aredescribed above; t and r are 0-100 independently; R₃, R₄, R₅, R₆, R₇,and R₈ are independently chosen from H; halide; C₁-C₈ alkyl; C₂-C₈ aryl,alkenyl, alkynyl, ether, ester, amine or amide, which is optionallysubstituted by one or more halide, CN, NR₁R₂, CF₃, OR₁, Aryl,heterocycle, S(O)R₁, SO₂R₁, —CO₂H, —SO₃H, —OR₁, —CO₂R₁, —CONR₁,—PO₂R₁R₂, —PO₃H or P(O)R₁R₂R₃; K is NR₁, —SS—, —C(═O)—, —C(═O)NH—,—C(═O)O—, —C═NH—O—, —C═N—NH—, —C(═O)NH—NH—, O, S, Se, B, Het(heterocyclic or heteroaromatic ring having C₃-C₈), or a peptidecontaining 1-20 amino acids.
 8. The side chain-linkaged compoundaccording to claim 1 having one of the following structures:

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers: wherein X₁,X₂, and X₃ are independently O, S, NH, NHNH, or CH₂; Z₂ and Z₃ areindependently NH, O, or S; p, p₁, p₂, and p₃ are independently 1-100; q₁and q₂ are independently selected from 0-24; L_(v3) is a leaving groupselected from F, Cl, Br, I, nitrophenol; N-hydroxysuccinimide (NHS);phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol;difluorophenol; monofluorophenol; pentachlorophenol; triflate;imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxy benzotriazole;tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3′ sulfonate, anhydrideformed by an acid itself, or formed with another anhydride: acetylanhydride, formyl anhydride; or an intermediate molecule generated witha condensation reagent for peptide coupling reactions or for Mitsunobureactions; Aa is natural or unnatural amino acid; r is 0-12; (Aa)_(r) isa peptide containing the same or different sequence of amino acids whenr>2; r=0 means (Aa)_(r) absent; R₂₅ and R₂₅′ are independently selectedfrom H; HC(O), CH₃C(O), CH₃C(NH), C₁-C₁₈ alkyl, C₁-C₁₈ alkyl,alkyl-Y₁—SO₃H, C₁-C₁₈ alkyl-Y₁—PO₃H₂, C₁-C₁₈ alkyl-Y₁—CO₂H, C₁-C₁₈alkyl-Y₁—N⁺R₁′R₂′R₃′R⁴′, C₁-C₁₈ alkyl-Y₁—CONH₂, C₂-C₁₈ alkylene, C₂-C₁₈ester, C₂-C₁₈ ether, C₂-C₁₈ amine, C₂-C₁₈ alkyl carboxylamide, C₃-C₁₈Aryl, C₃-C₁₈ cyclic alkyl, C₃-C₁₈ heterocyclic, 1-24 amino acids; C₂-C₁₈lipid, a C₂-C₁₈ fatty acid or a C₂-C₁₈ fatty ammonium lipid; and m is0-20.